Human gene critical to fertility

ABSTRACT

Disclosed herein are novel nucleic acid and protein sequences that are essential to fertility. In particular, human Mater cDNA and protein sequences are provided. Functional MATER is required for female fertility; zygotes that arise from Mater null oocytes do not progress beyond the two-cell stage. Methods are described for using Mater molecules in diagnoses, prognosis, and treatment of infertility and reduced fertility. Also provided are methods for using MATER as a contraceptive agent. The disclosure also describes compounds involved in such methods, and the identification of such compounds.

CROSS REFERENCE TO RELATED APPLICATIONS

This is the U.S. National Stage of International Application No.PCT/US01/10981, filed Apr. 4, 2001 (published in English under PCTArticle 21(2)), which in turn claims the benefit of U.S. ProvisionalApplication No. 60/241,510, filed Oct. 18, 2000. Both applications areincorporated herein in their entirety.

FIELD

The present disclosure is generally related to fertility, including themechanisms controlling it, diseases that arise from defects in suchmechanisms, and methods of influencing (either inhibiting or enhancing)fertility.

BACKGROUND

Premature ovarian failure (POF) is a term used to describe certain typesof infertility in women. As many as 1% of all women in the United Statesare thought to be afflicted with POF, which manifests as menopausal-typesymptoms, including infertility, in women under the age of 40. Manydifferent diseases and conditions can cause POF, including underlyingchromosomal defects (e.g., X-chromosome fragility), chemotherapy, orradiation treatment. Autoimmunity is a well-established mechanism ofpremature ovarian failure (see Yan et al., J Womens Health Gend BasedMed. 9:275–87, 2000; and Kalantaridou & Nelson, J Am Med Womens Assoc.53:18–20, 1998). In autoimmune infertility, a woman's ovaries areattacked by cells of her own immune system, leading to a condition knownas autoimmune oophoritis (inflammation of the ovary). Autoimmune diseasecan develop in response to a single inciting antigen and then spread toinvolve other antigenic molecules of the same organ (Kaufman, Nature366:69–72, 1993). Therefore, identifying the autoantigen target in anorgan-specific autoimmune disease is essential to understanding itspathogenesis.

An experimental animal (mouse) model has been used to gain insight intothe mechanisms of human autoimmune oophoritis. Removal of the thymus(thymectomy) in neonatal mice (about three days old) inducesexperimental autoimmune oophoritis in certain strains of mice (Taguchiet al., Clin Exp Immunol. 42:324–331, 1980). This experimentally inducedcondition leads to the production of high levels of anti-ooplasmantibodies and sterility, accompanied by follicular degeneration; theprogression of the condition appears to closely parallel humanautoimmune oophoritis (Kalantaridou & Nelson, J Am Med Womens Assoc.53:18–20, 1998).

Maternal products control the developmental program until embryonicgenome activation takes place. Maternal effect genes that are importantin early embryonic development have been well documented in Drosophilaand Xenopus (Morisato & Anderson, Annu. Rev. Genet 29:371–399, 1995;Newport & Kirschner, Cell 30:687–696, 1982), but their presence has onlybeen inferred in mammals (Gardner, Hum. Reprod Update 2:1–27, 1999). Inmice, embryonic transcription is first detected in the late 1-cellzygote stage and is required for development beyond the 2-cell stage(Schultz, Bioessays 15:531–538, 1993; Flach et al, EMBO J. 1:681–686,1982; Latham et al., Mol. Reprod Dev. 35:140–150, 1993). The factorsgoverning this transition from the maternal to the embryonic genome areunknown.

A critical transition in development occurs with the switch fromdependence on proteins stored in the egg to those that result fromactivation of the embryonic genome. This shift which occurs at thetwo-cell stage in mice is dependent on maternal factors. Genetranscription and protein translation are active during murine oogenesisand RNAs and proteins accumulate within oocytes. However, germ cellsbecomes transcriptionally inactive late in oogenesis and much of thematernal RNA is degraded during meiotic maturation and ovulation of theegg into the oviduct. Thus, few maternal gene products persist past thetwo-cell embryo stage and none have been demonstrated directly to affectearly development (Schultz, Bioessays 15, 531–538, 1993; Gardner, Hum.Reprod. Update 2, 3–27, 1996).

SUMMARY OF THE DISCLOSURE

Described herein is the human MATER protein, an approximately 135 kDa(predicted estimated molecular weight) cytoplasmic protein expressed inmammalian oocytes that is required for female fertility. Zygotes thatarise from a MATER null oocyte do not progress beyond the two-cellstage.

Some embodiments are an isolated human MATER protein predicted to havean estimated molecular weight of about 125 kDa to about 135 kDa, in someembodiments more particularly about 134.2 kDa. For instance; thisestimated molecular weight may be obtained by SDS-polyacrylamide gelelectrophoresis and Western blotting, for instance using an antibodyraised against a C-terminal peptide of murine MATER (e.g., residues 1093to 1111 of SEQ ID NO: 6). MATER proteins include an amino acid sequenceas set forth in SEQ ID NOs: 2 or 4 or 24, or a sequence having at least65% sequence identity with SEQ ID NOs: 2 or 4 or 24. Certain specificexamples of such proteins may contain one or more conservative variantswithin such sequences. The provided human MATER proteins have MATERprotein biological activity, for instance in that they can complement aMater null phenotype.

In certain embodiments, the human MATER protein is an autoantigenassociated with autoimmune infertility. The protein may be expressed inoocyte cytoplasm, for instance the oocyte cytoplasm of a mammal such asa human.

One specific embodiment is thus an isolated human MATER proteinpredicted to have an estimated molecular weight of about 135 kDa,wherein the human MATER protein comprises amino acid sequences as setforth in SEQ ID NO: 2 and/or SEQ ID NO: 4 and/or SEQ ID NO: 24, theprotein is an oocyte cytoplasm-specific autoantigen associated withautoimmune infertility, and it can complement a Mater null phenotype bypermitting progression of an embryo beyond the two-cell stage.

Further embodiments are isolated nucleic acid molecules that encode sucha MATER protein. Examples of such nucleic acid molecules include asequence as set forth in SEQ ID NOs: 1 or 3 or 23, or a sequence havingat least 82% sequence identity with SEQ ID NOs: 1 or 3 or 23. Certainexamples of such nucleic acid molecules may contain one or moreconservative variants within such sequences. Certain examples hybridizeswith a nucleic acid probe that includes the sequence shown in SEQ ID NO:1 or SEQ ID NO: 3 or SEQ ID NO: 23 under wash conditions of 55° C.,0.2×SSC and 0.1% SDS, or under wash conditions of 50° C., 2×SSC, 0.1%SDS.

Also provided are recombinant nucleic acid molecules that include apromoter sequence operably linked to a MATER nucleic acid molecule andcells transformed with such a recombinant nucleic acid molecule.

Methods of detecting a biological condition associated with an abnormalMater nucleic acid or an abnormal MATER expression or an autoimmuneresponse to MATER in a subject are also provided. Examples of such abiological condition include infertility (such as autoimmuneinfertility) or reduced fertility, or an increased susceptibility toinfertility or reduced fertility. Such methods can involve detecting anabnormal Mater nucleic acid or an abnormal Mater expression or theautoimmune response to MATER in the subject. In specific examples ofsuch methods, the abnormal Mater nucleic acid or abnormal Materexpression includes an alteration in cellular level of Mater nucleicacid or MATER protein, in comparison to a normal level. The abnormalMater expression may include an increased or decreased expression ofMater in a subject.

Specific provided methods involve determining whether the subject hascirculating autoantibodies that recognize an epitope of a MATER protein,wherein presence of such autoantibodies indicates the infertility orreduced fertility of the subject, or an increased susceptibility of thesubject to infertility or reduced fertility. Other specific methodsinvolve reacting at least one Mater molecule contained in a clinicalsample from the subject with a reagent that includes a Mater specificbinding agent (such as an oligonucleotide or a MATER protein specificbinding agent (e.g., an antibody or functional fragment thereof), toform a Mater:agent complex.

Also provided are methods for detecting a predisposition to infertilityor reduced fertility or for presymptomatic screening of an individualfor infertility or reduced fertility.

Specific methods of detecting a biological condition provided hereininvolve in vitro amplifying a Mater nucleic acid prior to detecting theabnormal Mater nucleic acid. Amplification can be performed, forinstance, using at least one oligonucleotide primer derived from a MATERprotein encoding sequence. Examples of such oligonucleotides may includeat least 15, for instance at least 20 or at least 23, contiguousnucleotides from SEQ ID NO: 1 or SEQ ID NO: 3 or SEQ ID NO: 23.

Also provided are oligonucleotide primers used in such methods,recombinant DNA vectors that contain such nucleic acid molecules, andrecombinant nucleic acid molecules that include a promoter sequenceoperably linked (in sense or antisense orientation) to such a nucleicacid molecule. Such vectors and recombinant nucleic acid molecules canbe transformed into cells or animals (e.g., non-human animals); suchtransformed cells and animals are also provided.

In certain examples of the provided methods, the Mater molecule is aMATER encoding sequence. In some of such examples, a Mater:agent complexis detected by nucleotide hybridization, for instance where the agent isa labeled nucleotide probe. Such probes may include a sequence as shownin SEQ ID NOs: 1 or 3 or 23, fragments of these sequences (for instance,fragments of 23 or more nucleotides). Other probes may contain asequence that shares at least 70% sequence identity with a sequence asshown in SEQ ID NOs: 1 or 3 or 23. Such nucleotide probes are alsoprovided.

In other examples of the provided methods, the Mater molecule is a MATERprotein, which may contain the sequence of SEQ ID NOs: 2 or 4 or 24, asequence sharing at least 65% sequence identity with SEQ ID NOs: 2 or 4or 24, or conservative variants thereof. In some of such examples,Mater:agent complexes are detected by Western blot assay or by ELISA. Incertain methods provided, the Mater-specific binding agent is aMATER-specific antibody or a functional fragment thereof, for instance apolyclonal or monoclonal antibody. In specific examples, the antibodyrecognizes a peptide that includes the sequence of SEQ ID NOs: 2 or 4 or24, or an antigenic fragment of one of these peptides.

Also provided herein are kits, including kits for detecting an over- orunder-abundance of MATER protein or Mater nucleic acid (for instance, ina sample from a subject, such as a mammal), which kits include a MATERprotein specific binding agent (such as an antibody or a functionalfragment thereof). In certain examples, the agent is capable ofspecifically binding to an epitope within the amino acid sequence shownin SEQ ID NOs: 2 or 4 or 24, an amino acid sequences that differ fromthese by one or more conservative amino acid substitutions, an aminoacid sequences having at least 65% sequence identity to these sequences,or an antigenic fragment of any of these sequences. Particular examplesof such kits further include a means for detecting binding of the MATERprotein binding agent to a MATER polypeptide. In certain examples ofthese kits, the overabundance or underabundance of MATER protein orMater nucleic acid that is detected results in altered infertility.

Other provided kits are for detecting a genetic mutation (e.g., amutation in a Mater sequence) in a sample of nucleic acid. Such kits mayinclude an oligonucleotide capable of specifically hybridizing with aMater nucleic acid (which may be provided in a first container), and afluorescent labeled nucleic acid probe (for instance, of about 5 to 500nucleotides) that is fully complementary to the oligonucleotide (whichmay be provided in a second container).

Specific kit embodiments provided herein are for determining whether ornot a subject has a biological condition associated with an abnormalMater expression by detecting an underabundance of MATER protein in asample of tissue and/or body fluids from the subject. Such kits includean antibody specific for MATER protein and instructions for using thekit. Such instructions may indicate steps for performing a method todetect the presence of MATER protein in the sample (for instance, usinga method described herein); and analyzing data generated by the method,wherein the instructions indicate that underabundance of MATER proteinin the sample indicates that the individual has or is predisposed to thebiological condition. Specific examples of such kits further include adetectable antibody that binds to the MATER protein specific antibody.

Other specific kit embodiments include a MATER protein specific antibody(which may be provided in a container), a negative control sample whichmay be provided in a container), instructions for using the kit. Suchinstructions may indicating steps for performing a test assay to detecta quantity of MATER protein in a test sample of tissue and/or bodilyfluid from the subject (such as a test assay provided herein),performing a negative control assay to detect a quantity of MATERprotein in the negative control sample; and comparing data generated bythe test assay and negative control assay. In specific examples of suchkits, the instructions indicate that a quantity of MATER protein in thetest sample that is less than the quantity of MATER protein in thenegative control sample indicates that the subject has the biologicalcondition. Some of such kits further include a detectable antibody thatbinds to the antibody specific for MATER protein (which may be providedin a separate container).

Also provided are methods of modifying the level of expression of aMATER protein in an subject, for instance by expressing in the subject arecombinant genetic construct that includes a promoter operably linked(in either sense or antisense orientation) to a nucleic acid moleculethat includes at least 23 consecutive nucleotides of SEQ ID NO: 1, SEQID NO: 3, SEQ ID NO: 23 or a sequence at least 70% identical to SEQ IDNO: 1, SEQ ID NO: 3, or SEQ ID NO: 23. In particular examples,expression of the nucleic acid molecule changes expression of the MATERprotein.

A further embodiment provides methods of screening for a compound usefulin influencing MATER-mediated fertility in a mammal. Such methodsinvolve determining if a test compound (for instance, when it is appliedto a test cell) binds to or interacts with a MATER protein, such as ahuman MATER protein, or a variant or fragment thereof, and selecting acompound that so binds. In particular examples of such methods, bindingof the compound inhibits a MATER protein biological activity. Alsoencompassed herein are compounds selected by these methods, for instancesuch compounds for use as contraceptives or fertility enhancers.

The foregoing and other features and advantages of the invention willbecome more apparent from the following detailed description of severalembodiments, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows two digitized gels, and a series of digitized micrographs,detailing the developmental expression of murine MATER.

FIG. 1A is a digitized image of a Northern blots showing RNAseprotection of Mater and ZP3 transcripts. RNase protection assays wereperformed using total RNA from 50 oocytes, eggs or embryos. ³²P-labeledantisense probes and protected fragment lengths for Mater and ZP3 were180/139 nt and 257/205 nt, respectively.

FIG. 1B is a digitized image of a Western blot, showing MATER protein(approximately 125 kD) amounts in the indicated murine cell types. MATERprotein was assayed in 25 oocytes, eggs or embryos by immunoblottingwith monospecific antisera to MATER.

FIG. 1C is a series of digitized micrographs, showing the cellularlocalization of MATER protein. Protein localization was determined usingfluorescent labeled antibody, by imaging oocytes, eggs and embryos withlaser-scanning confocal microscopy alone (upper images) or aftersuperimposition on a Nomarski image (lower images). Scale bar, 50 μm.

FIG. 2 shows the characterization of targeted Mater locus disruptions.

FIG. 2A is a schematic representation of the normal murine Mater allele(upper), the targeting construct (middle) and the null allele (bottom).The 5′ and 3′ probes used to assess targeting were outside the region ofDNA homology.

FIG. 2B is a series of digitized Northern blots, showing that the Maternull allele was detected in XbaI digested ES cell DNA as a 3.5 kbfragment with the 5′ (left) or as a 4.0 kb fragment with the 3′ (middle)and neo (right) probes.

FIG. 2C is a pair of digitized Northern blots, showing that Matertranscripts were detected in normal and heterozygous (half the abundanceof normal), but not in homozygous null ovaries (left panel). ZP3transcripts (control) were present in all three genotypes (right panel).

FIG. 3 is a series of digitized micrographs and corresponding bargraphs, showing the in vivo development defects of embryos derived fromMater null female mice.

After gonadotrophin induced ovulation, female mice were mated withnormal males and oviducts from normal (FIGS. 3A, 3D, 3G, and 3J) orMater null (FIGS. 3B, 3E, 3H, and 3K) females were flushed one (FIGS. 3Aand 3B), two (FIGS. 3D and 3E), three (FIGS. 3G and 3H) and four (FIGS.3J and 3K) days later. The unfixed embryos were photographed usingNomarski optics. The arrows point to pronuclei in 1-cell zygotes (FIGS.3A and 3B). Scale bar, 50 μm.

The bar graphs indicate developmental progress of the average number ofembryos derived from Mater null (▪) and normal (□) females at one (FIG.3C), two (FIG. 3F), three (FIG. 3I), and four (FIG. 3L) days aftermating. Each bar represents the average of 4–5 experiments±s.e.m.

FIG. 4 shows that de novo transcription and translation occurs in murineembryos lacking Mater.

FIGS. 4A–4F show digitized micrograph images of murine embryos. Newlysynthesized RNA was measured by BrUTP incorporation into the nucleus ofone (FIGS. 4A and 4B) and 2-cell (FIGS. 4C and 4D) embryos derived fromMater null (FIGS. 4A and 4C) and normal (FIGS. 4B and 4D) females usinglaser-scanning confocal microscopy and a monoclonal antibody to BrUTP.

FIG. 4G is a bar graph, showing the quantity of BrUTP incorporated in2-cell embryos with (+) and without (−) Mater using arbitraryfluorescence units after subtraction of that obtained with uninjectedcontrols (FIGS. 4E and 4F). Scale bar, 50 μM.

FIG. 4H is a pair of digitized northern blots, showing RNase protectionassays of 28S ribosomal RNA using total RNA from 50 growing oocytes,eggs and 2-cell embryos isolated from females with (+) and without (−)Mater. ³²P-labeled antisense probes and protected fragment lengths for28S-rRNA were 153 nt and 115 nt, respectively.

FIG. 4I is a series of digitized fluorographs, showing de novo proteinsynthesis in 1-cell zygotes (left panel) and 2-cell embryos (middle andright panels) that do (+) or do not (−) contain Mater. Each lanecontained proteins from 10 embryos that were dissolved in sample bufferdirectly (left and middle panels) or after partial purificationextraction of TRC (65–75 kDa) and p35 (right panels).

FIG. 5 shows ovarian histology, eggs and the number of eggs producedfrom Mater null females. The number of ovulated eggs and theirmorphology indicating maturation or degeneration were not remarkablydifferent between normal and Mater null mice.

FIGS. 5A and 5B are digitized images of ovarian slices from a 4 week old(FIG. 5A) and 8 week old (FIG. 5B) Mater null mouse. The 4 week oldMater null ovarian histology (FIG. 5A) displays different stages ofnormal ovarian follicles with oogenesis, which are indistinguishablefrom normal ovarian histology. In the 8 week old sample (FIG. 5B), thereare a number of corpora lutea indicating normal spontaneous ovulation,similar to follicular luteinization in the wild-type mouse ovary.

FIG. 5C is a digitized photograph of ovulated eggs from the Mater nullmice, produced in response to exogenous PMSG and hCG. Morphology appearssimilar to that of ovulated eggs from wild-type mice.

FIG. 5D is a bar graphs showing the numbers (mean±s.e.m.) of theovulated eggs from the Mater null and wild-type mice.

FIG. 6 is a digitized image comparing the gross morphological appearanceof the uterus and ovaries of normal and Mater null mice. Ovaries anduteri if Mater null female mice (right) were indistinguishable from theovaries and uteri from the wild-type female mice (left).

FIG. 7 is a series of digitized micrographs, showing oocyte-specificexpression of Mater transcripts in human by in situ hybridization. Both[³⁵S]-labeled antisense and sense probes were synthesized by in vitrotranscription using the cloned human Mater cDNA as a template. Thefrozen human ovarian sections were hybridized with the radiolabeledsense (FIGS. 7A and 7C) and antisense (FIGS. 7B and 7D) probes. Theslides were stained with hematoxylin and eosin. For each probe,bright-field (FIGS. 7A and 7B) and dark-field (FIGS. 7C and 7D) imagesare shown.

FIG. 8 is a pair of digitized micrographs, showing oocyte-specificexpression of human MATER protein. Frozen human ovarian sections wereincubated with rabbit antisera (1:200) against a C-terminal peptide ofmouse MATER protein, and FITC-conjugated goat anti-rabbit IgG antiserawere used as the second antibody to detect human MATER protein in theoocyte (FIG. 8A). The corresponding phase contrast image is shown inFIG. 8B.

FIG. 9 is an alignment of the amino acid sequence of human MATER (SEQ IDNO: 24) and murine MATER (SEQ ID NO: 6) proteins. Identical amino acidsare identified; similar amino acids are indicated with a plus (+)symbol.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. In the accompanying sequence listing:

SEQ ID NO: 1 shows the nucleic acid sequence of human Mater cDNAfragment 1.

SEQ ID NO: 2 shows the amino acid sequence of the human MATER peptideencoded by fragment 1 (SEQ ID NO: 1).

SEQ ID NO: 3 shows the nucleic acid sequence of human Mater cDNAfragment 2.

SEQ ID NO: 4 shows the amino acid sequence of the human MATER peptideencoded by fragment 2 (SEQ ID No: 3).

SEQ ID NO: 5 shows the nucleic acid sequence of murine Mater cDNA(GenBank Accession number NM_(—)011860.1 and AF074018; individual exonsare also listed in AF143559–AF143573).

SEQ ID NO: 6 shows the amino acid sequence of the murine MATER protein(GenBank Accession number NP_(—)035990).

SEQ ID NOs: 7–22 shows several synthetic oligonucleotides useful asprobes and/or primers.

SEQ ID NO: 23 shows the nucleic acid sequence of the human Mater cDNA,and the amino acid sequence so encoded.

SEQ ID NO: 24 shows the amino acid sequence of the human MATER protein.

DETAILED DESCRIPTION OF THE DISCLOSURE

I. Abbreviations

ES: embryonic stem

Mater: Maternal Antigen That Embryos Require

OP1: Ooplasm-specific Protein 1

POF: Premature Ovarian Failure

TRC: Transcription Related Complex

ZP3: zona pellucida glycoprotein 3

II. Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of theinvention, the following explanations of specific terms are provided:

Abnormal: Deviation from normal characteristics. Normal characteristicscan be found in a control, a standard for a population, etc. Forinstance, where the abnormal condition is an autoimmune diseasecondition, such as autoimmune infertility, sources of normalcharacteristics might include an individual who is not suffering fromthe autoimmune disorder, a population standard of individuals believednot to be suffering from the disease, etc.

In some examples, abnormal may refer to a condition that is associatedwith a disease. The term “associated with” includes an increased risk ofdeveloping the disease as well as the disease itself. For instance, acertain abnormality (such as an abnormality in an Mater nucleic acid orMATER protein expression) can be described as being associated with thebiological conditions of altered (e.g., reduced) fertility and tendencyto develop autoimmune infertility.

An abnormal nucleic acid, such as an abnormal Mater nucleic acid, is onethat is different in some manner to a normal (wildtype) nucleic acid.Such abnormality includes but is not necessarily limited to: (1) amutation in the nucleic acid (such as a point mutation (e.g., a singlenucleotide polymorphism) or short deletion or duplication of a few toseveral nucleotides); (2) a mutation in the control sequence(s)associated with that nucleic acid such that replication or expression ofthe nucleic acid is altered (such as the functional inactivation of apromoter); (3) a decrease in the amount or copy number of the nucleicacid in a cell or other biological sample (such as a deletion of thenucleic acid, either through selective gene loss or by the loss of alarger section of a chromosome or under expression of the mRNA); and (4)an increase in the amount or copy number of the nucleic acid in a cellor sample (such as a genomic amplification of part or all of the nucleicacid or the overexpression of an mRNA), each compared to a control orstandard. It will be understood that these types of abnormalities canco-exist in the same nucleic acid or in the same cell or sample; forinstance, a genomic-amplified nucleic acid sequence may also contain oneor more point mutations. In addition, it is understood that anabnormality in a nucleic acid may be associated with, and in fact maycause, an abnormality in expression of the corresponding protein.

Abnormal protein expression, such as abnormal MATER protein expression,refers to expression of a protein that is in some manner different toexpression of the protein in a normal (wildtype) situation. Thisincludes but is not necessarily limited to: (1) a mutation in theprotein such that one or more of the amino acid residues is different;(2) a short deletion or addition of one or a few amino acid residues tothe sequence of the protein; (3) a longer deletion or addition of aminoacid residues, such that an entire protein domain or sub-domain isremoved or added; (4) expression of an increased amount of the protein,compared to a control or standard amount; (5) expression of an decreasedamount of the protein, compared to a control or standard amount; (6)alteration of the subcellular localization or targeting of the protein;(7) alteration of the temporally regulated expression of the protein(such that the protein is expressed when it normally would not be, oralternatively is not expressed when it normally would be); and (8)alteration of the localized (e.g., organ or tissue specific) expressionof the protein (such that the protein is not expressed where it wouldnormally be expressed or is expressed where it normally would not beexpressed), each compared to a control or standard.

Controls or standards appropriate for comparison to a sample, for thedetermination of abnormality, include samples believed to be normal aswell as laboratory values, even though possibly arbitrarily set, keepingin mind that such values may vary from laboratory to laboratory.Laboratory standards and values may be set based on a known ordetermined population value and may be supplied in the format of a graphor table that permits easy comparison of measured, experimentallydetermined values.

Antisense, Sense, and Antigene: Double-stranded DNA (dsDNA) has twostrands, a 5′->3′ strand, referred to as the plus strand, and a 3′->5′strand (the reverse complement), referred to as the minus strand.Because RNA polymerase adds nucleic acids in a 5′->3′ direction, theminus strand of the DNA serves as the template for the RNA duringtranscription. Thus, the RNA formed will have a sequence complementaryto the minus strand and identical to the plus strand (except that U issubstituted for T).

Antisense molecules are molecules that are specifically hybridizable orspecifically complementary to either RNA or the plus strand of DNA.Sense molecules are molecules that are specifically hybridizable orspecifically complementary to the minus strand of DNA. Antigenemolecules are either antisense or sense molecules directed to a dsDNAtarget.

Binding or stable binding: An oligonucleotide binds or stably binds to atarget nucleic acid if a sufficient amount of the oligonucleotide formsbase pairs or is hybridized to its target nucleic acid, to permitdetection of that binding. Binding can be detected by either physical orfunctional properties of the target:oligonucleotide complex. Bindingbetween a target and an oligonucleotide can be detected by any procedureknown to one skilled in the art, including both functional and physicalbinding assays. Binding may be detected functionally by determiningwhether binding has an observable effect upon a biosynthetic processsuch as expression of a gene, DNA replication, transcription,translation, and the like.

Physical methods of detecting the binding of complementary strands ofDNA or RNA are well known in the art, and include such methods as DNaseI or chemical footprinting, gel shift and affinity cleavage assays,Northern blotting, dot blotting and light absorption detectionprocedures. For example, one method that is widely used, because it isso simple and reliable, involves observing a change in light absorptionof a solution containing an oligonucleotide (or an analog) and a targetnucleic acid at 220 to 300 nm as the temperature is slowly increased. Ifthe oligonucleotide or analog has bound to its target, there is a suddenincrease in absorption at a characteristic temperature as theoligonucleotide (or analog) and target disassociate from each other, ormelt.

The binding between an oligomer and its target nucleic acid isfrequently characterized by the temperature (T_(m)) at which 50% of thetarget sequence remains hybridized to a perfectly matched probe orcomplementary strand. A higher (T_(m)) means a stronger or more stablecomplex relative to a complex with a lower (T_(m)).

cDNA (complementary DNA): A piece of DNA lacking internal, non-codingsegments (introns) and transcriptional regulatory sequences. cDNA mayalso contain untranslated regions (UTRs) that are responsible fortranslational control in the corresponding RNA molecule. cDNA is usuallysynthesized in the laboratory by reverse transcription from messengerRNA extracted from cells.

DNA (deoxyribonucleic acid): DNA is a long chain polymer which comprisesthe genetic material of most living organisms (some viruses have genescomprising ribonucleic acid (RNA)). The repeating units in DNA polymersare four different nucleotides, each of which comprises one of the fourbases, adenine (A), guanine (G), cytosine (C), and thymine (T) bound toa deoxyribose sugar to which a phosphate group is attached. Triplets ofnucleotides (referred to as codons) code for each amino acid in apolypeptide, or for a stop signal. The term codon is also used for thecorresponding (and complementary) sequences of three nucleotides in themRNA into which the DNA sequence is transcribed.

Unless otherwise specified, any reference to a DNA molecule is intendedto include the reverse complement of that DNA molecule. Except wheresingle-strandedness is required by the text herein, DNA molecules,though written to depict only a single strand, encompass both strands ofa double-stranded DNA molecule. Thus, a reference to the nucleic acidmolecule that encodes a specific protein, or a fragment thereof,encompasses both the sense strand and its reverse complement. Thus, forinstance, it is appropriate to generate probes or primers from thereverse complement sequence of the disclosed nucleic acid molecules.

Deletion: The removal of a sequence of DNA, the regions on either sideof the removed sequence being joined together.

Gene amplification or genomic amplification: An increase in the copynumber of a gene or a fragment or region of a gene or associated 5′ or3′ region, as compared to the copy number in normal tissue. An exampleof a genomic amplification is an increase in the copy number of anoncogene. A “gene deletion” is a deletion of one or more nucleic acidsnormally present in a gene sequence and, in extreme examples, caninclude deletions of entire genes or even portions of chromosomes.

Hybridization: Oligonucleotides and their analogs hybridize by hydrogenbonding, which includes Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary bases. Generally, nucleic acidconsists of nitrogenous bases that are either pyrimidines (cytosine (C),uracil (U), and thymine (T)) or purines (adenine (A) and guanine (G)).These nitrogenous bases form hydrogen bonds between a pyrimidine and apurine, and the bonding of the pyrimidine to the purine is referred toas “base pairing.” More specifically, A will hydrogen bond to T or U,and G will bond to C. “Complementary” refers to the base pairing thatoccurs between to distinct nucleic acid sequences or two distinctregions of the same nucleic acid sequence.

“Specifically hybridizable” and “specifically complementary” are termsthat indicate a sufficient degree of complementarity such that stableand specific binding occurs between the oligonucleotide (or its analog)and the DNA or RNA target. The oligonucleotide or oligonucleotide analogneed not be 100% complementary to its target sequence to be specificallyhybridizable. An oligonucleotide or analog is specifically hybridizablewhen binding of the oligonucleotide or analog to the target DNA or RNAmolecule interferes with the normal function of the target DNA or RNA,and there is a sufficient degree of complementarity to avoidnon-specific binding of the oligonucleotide or analog to non-targetsequences under conditions where specific binding is desired, forexample under physiological conditions in the case of in vivo assays orsystems. Such binding is referred to as specific hybridization.

Hybridization conditions resulting in particular degrees of stringencywill vary depending upon the nature of the hybridization method ofchoice and the composition and length of the hybridizing nucleic acidsequences. Generally, the temperature of hybridization and the ionicstrength (especially the Na⁺ concentration) of the hybridization bufferwill determine the stringency of hybridization, though waste times alsoinfluence stringency. Calculations regarding hybridization conditionsrequired for attaining particular degrees of stringency are discussed bySambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2nd ed.,vol. 1–3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1989, chapters 9 and 11, herein incorporated by reference.

For present purposes, “stringent conditions” encompass conditions underwhich hybridization will only occur if there is less than 25% mismatchbetween the hybridization molecule and the target sequence. “Stringentconditions” may be broken down into particular levels of stringency formore precise definition. Thus, as used herein, “moderate stringency”conditions are those under which molecules with more than 25% sequencemismatch will not hybridize; conditions of “medium stringency” are thoseunder which molecules with more than 15% mismatch will not hybridize,and conditions of “high stringency” are those under which sequences withmore than 10% mismatch will not hybridize. Conditions of “very highstringency” are those under which sequences with more than 6% mismatchwill not hybridize.

In vitro amplification: Techniques that increases the number of copiesof a nucleic acid molecule in a sample or specimen. An example ofamplification is the polymerase chain reaction, in which a biologicalsample collected from a subject is contacted with a pair ofoligonucleotide primers, under conditions that allow for thehybridization of the primers to nucleic acid template in the sample. Theprimers are extended under suitable conditions, dissociated from thetemplate, and then re-annealed, extended, and dissociated to amplify thenumber of copies of the nucleic acid. The product of in vitroamplification may be characterized by electrophoresis, restrictionendonuclease cleavage patterns, oligonucleotide hybridization orligation, and/or nucleic acid sequencing, using standard techniques.Other examples of in vitro amplification techniques include stranddisplacement amplification (see U.S. Pat. No. 5,744,311);transcription-free isothermal amplification (see U.S. Pat. No.6,033,881); repair chain reaction amplification (see WO 90/01069);ligase chain reaction amplification (see EP-A-320 308); gap fillingligase chain reaction amplification (see U.S. Pat. No. 5,427,930);coupled ligase detection and PCR (see U.S. Pat. No. 6,027,889); andNASBA™ RNA transcription-free amplification (see U.S. Pat. No.6,025,134).

Injectable composition: A pharmaceutically acceptable fluid compositionincluding at least one active ingredient. The active ingredient isusually dissolved or suspended in a physiologically acceptable carrier,and the composition can additionally include minor amounts of one ormore non-toxic auxiliary substances, such as emulsifying agents,preservatives, and pH buffering agents and the like. Such injectablecompositions that are useful for use with the provided nucleotides andproteins are conventional; appropriate formulations are well known inthe art.

Isolated: An “isolated” biological component (such as a nucleic acidmolecule, protein or organelle) has been substantially separated orpurified away from other biological components in the cell of theorganism in which the component naturally occurs, i.e., otherchromosomal and extra-chromosomal DNA and RNA, proteins and organelles.Nucleic acids and proteins that have been “isolated” include nucleicacids and proteins purified by standard purification methods. The termalso embraces nucleic acids and proteins prepared by recombinantexpression in a host cell as well as chemically synthesized nucleicacids.

MATER protein: A cytosol-localized protein (SEQ ID NO: 24) ofapproximately 125 to approximately 135 kDa (estimated molecular weightbased on gel mobility) that is essential for female fertility. Theacronym stands for Maternal Antigen That Embryos Require. Mater is asingle-copy gene found on human Chromosome 19. Zygotes that arise fromMater null (Mater⁻¹) oocytes do not progress beyond the two-cell stage.Thus, Mater represents a novel maternal effect gene that is required forembryonic survival and early development in mammals.

Proteins can be identified as MATER proteins by comparing their activityand other physical characteristics to a prototypical MATER protein, suchas the human or murine MATER protein. MATER protein biological activitycan be described in terms of the ability of a protein to complement(substantially replace the lost function in) a Mater null mutant. Theability of a protein to complement a Mater mutant may be readilydetermined by introducing the gene encoding the protein into a Matermutant animal system (such as the Mater null mice described herein)using standard methods. If the encoded protein has MATER proteinbiological activity, this will be manifested as a proportion of thetransgenic progeny animals having a relatively wild-type phenotype forthose characteristics linked to the Mater mutant (e.g., infertility ofMater null females due to failure of Mater null oocytes to progressbeyond the two-cell stage).

Other MATER protein physical characteristics that can be examined whenevaluating a hypothetical MATER protein include the molecular weight ofthe protein (approximately 125–135 kDa, see Example 1, though this valuemay vary somewhat from species to species), the subcellular localizationof the protein (human MATER is expressed in oocytes (Example 4)), and ispredicted to be specifically expressed in the cytoplasm and excludedfrom the nucleus, as occurs in mice (see Example 1), and the temporaland spatial regulation of the mRNA (Mater transcript is produced in thematuring oocyte, see Examples 1 and 3). Antibodies that recognize oneMATER protein (e.g., a murine MATER) may recognize a MATER protein fromanother species (e.g. human MATER) (see Example 4); thus, hypotheticalMATER proteins can be further examined and identified based onrecognition by anti-MATER antibodies produced against MATER proteinsfrom other species. The identity of the human MATER protein cantherefore be confirmed for instance by immunological identificationusing an antibody raised against the murine MATER protein, or an epitopeof that protein.

Nucleotide: “Nucleotide” includes, but is not limited to, a monomer thatincludes a base linked to a sugar, such as a pyrimidine, purine orsynthetic analogs thereof, or a base linked to an amino acid, as in apeptide nucleic acid (PNA). A nucleotide is one monomer in apolynucleotide. A nucleotide sequence refers to the sequence of bases ina polynucleotide.

Oligonucleotide: An oligonucleotide is a plurality of joined nucleotidesjoined by native phosphodiester bonds, between about 6 and about 300nucleotides in length. An oligonucleotide analog refers to moieties thatfunction similarly to oligonucleotides but have non-naturally occurringportions. For example, oligonucleotide analogs can contain non-naturallyoccurring portions, such as altered sugar moieties or inter-sugarlinkages, such as a phosphorothioate oligodeoxynucleotide. Functionalanalogs of naturally occurring polynucleotides can bind to RNA or DNA,and include peptide nucleic acid (PNA) molecules.

Particular oligonucleotides and oligonucleotide analogs can includelinear sequences up to about 200 nucleotides in length, for example asequence (such as DNA or RNA) that is at least 6 bases, for example atleast 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100 or even 200 bases long,or from about 6 to about 50 bases, for example about 10–25 bases, suchas 12, 15, 20, or 23 bases.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein-coding regions, in the samereading frame.

Open reading frame: A series of nucleotide triplets (codons) coding foramino acids without any internal termination codons. These sequences areusually translatable into a peptide.

Ortholog: Two nucleic acid or amino acid sequences are orthologs of eachother if they share a common ancestral sequence and diverged when aspecies carrying that ancestral sequence split into two species.Orthologous sequences are also homologous sequences.

Parenteral: Administered outside of the intestine, e.g. not via thealimentary tract. Generally, parenteral formulations are those that willbe administered through any possible mode except ingestion. This termespecially refers to injections, whether administered intravenously,intrathecally, intramuscularly, intraperitoneally, or subcutaneously,and various surface applications including intranasal, intradermal, andtopical application, for instance.

Peptide Nucleic Acid (PNA): An oligonucleotide analog with a backbonecomprised of monomers coupled by amide (peptide) bonds, such as aminoacid monomers joined by peptide bonds.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers useful with the compositions provided herein are conventional.Martin, Remington's Pharmaceutical Sciences, published by MackPublishing Co., Easton, Pa., 19th Edition, 1995, describes compositionsand formulations suitable for pharmaceutical delivery of the nucleotidesand proteins herein disclosed.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (e.g., powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically-neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate.

Polymorphism: Variant in a sequence of a gene. Polymorphisms can bethose variations (nucleotide sequence differences) that, while having adifferent nucleotide sequence, produce functionally equivalent geneproducts, such as those variations generally found between individuals,different ethnic groups, geographic locations. The term polymorphismalso encompasses variations that produce gene products with alteredfunction, i.e., variants in the gene sequence that lead to gene productsthat are not functionally equivalent. This term also encompassesvariations that produce no gene product, an inactive gene product, orincreased gene product. The term polymorphism may be usedinterchangeably with allele or mutation, unless context clearly dictatesotherwise.

Polymorphisms can be referred to, for instance, by the nucleotideposition at which the variation exists, by the change in amino acidsequence caused by the nucleotide variation, or by a change in someother characteristic of the nucleic acid molecule that is linked to thevariation (e.g., an alteration of a secondary structure such as astem-loop, or an alteration of the binding affinity of the nucleic acidfor associated molecules, such as polymerases, RNases, and so forth).

Probes and primers: Nucleic acid probes and primers can be readilyprepared based on the nucleic acid molecules provided as indicators ofdisease or disease progression. It is also appropriate to generateprobes and primers based on fragments or portions of these nucleic acidmolecules. Also appropriate are probes and primers specific for thereverse complement of these sequences, as well as probes and primers to5′ or 3′ regions.

A probe comprises an isolated nucleic acid attached to a detectablelabel or other reporter molecule. Typical labels include radioactiveisotopes, enzyme substrates, co-factors, ligands, chemiluminescent orfluorescent agents, haptens, and enzymes. Methods for labeling andguidance in the choice of labels appropriate for various purposes arediscussed, e.g. in Sambrook et al. (In Molecular Cloning: A LaboratoryManual, CSHL, New York, 1989) and Ausubel et al (In Current Protocols inMolecular Biology, John Wiley & Sons, New York, 1998).

Primers are short nucleic acid molecules, for instance DNAoligonucleotides 10 nucleotides or more in length. Longer DNAoligonucleotides may be about 15, 20, 25, 30 or 50 nucleotides or morein length. Primers can be annealed to a complementary target DNA strandby nucleic acid hybridization to form a hybrid between the primer andthe target DNA strand, and then the primer extended along the target DNAstrand by a DNA polymerase enzyme. Primer pairs can be used foramplification of a nucleic acid sequence, e.g., by the polymerase chainreaction (PCR) or other in vitro nucleic-acid amplification methodsknown in the art.

Methods for preparing and using nucleic acid probes and primers aredescribed, for example, in Sambrook et al. (In Molecular Cloning: ALaboratory Manual, CSHL, New York, 1989), Ausubel et al. (ed.) (InCurrent Protocols in Molecular Biology, John Wiley & Sons, New York,1998), and Innis et al. (PCR Protocols, A Guide to Methods andApplications, Academic Press, Inc., San Diego, Calif., 1990).Amplification primer pairs (for instance, for use with polymerase chainreaction amplification) can be derived from a known sequence such as theMater sequences described herein, for example, by using computerprograms intended for that purpose such as Primer (Version 0.5, © 1991,Whitehead Institute for Biomedical Research, Cambridge, Mass.).

One of ordinary skill in the art will appreciate that the specificity ofa particular probe or primer increases with its length. Thus, forexample, a primer comprising 30 consecutive nucleotides of a MATERprotein encoding nucleotide will anneal to a target sequence, such asanother homolog of the designated MATER protein, with a higherspecificity than a corresponding primer of only 15 nucleotides. Thus, inorder to obtain greater specificity, probes and primers can be selectedthat comprise at least 20, 23, 25, 30, 35, 40, 45, 50 or moreconsecutive nucleotides of a MATER protein-encoding nucleotidesequences.

Also provided are isolated nucleic acid molecules that comprisespecified lengths of the disclosed Mater nucleotide sequences. Suchmolecules may comprise at least 10, 15, 20, 23, 25, 30, 35, 40, 45 or 50or more consecutive nucleotides of these sequences or more, and may beobtained from any region of the disclosed sequences (e.g., a Maternucleic acid may be apportioned into halves or quarters based onsequence length, and isolated nucleic acid molecules may be derived fromthe first or second halves of the molecules, or any of the fourquarters, etc.). A Mater cDNA or other encoding sequence also can bedivided into smaller regions, e.g about eighths, sixteenths, twentieths,fiftieths and so forth, with similar effect.

Another mode of division is to select the 5′ (upstream) and/or 3′(downstream) region associated with a Mater gene.

Nucleic acid molecules may be selected that comprise at least 10, 15,20, 25, 30, 35, 40, 50 or 100 or more consecutive nucleotides of any ofthese or other portions of a human Mater nucleic acid molecule, such asthose disclosed herein, and associated flanking regions. Thus,representative nucleic acid molecules might comprise at least 10consecutive nucleotides of the human cDNA fragments show in SEQ ID NOs:1 and 2, or the full human MATER cDNA shown in SEQ ID NO: 24.

Protein: A biological molecule expressed by a gene or recombinant orsynthetic coding sequence and comprised of amino acids.

Purified: The term “purified” does not require absolute purity; rather,it is intended as a relative term. Thus, for example, a purified proteinpreparation is one in which the protein referred to is more pure thanthe protein in its natural environment within a cell or within aproduction reaction chamber (as appropriate).

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occurring or has a sequence that is made by anartificial combination of two otherwise separated segments of sequence.This artificial combination can be accomplished by chemical synthesisor, more commonly, by the artificial manipulation of isolated segmentsof nucleic acids, e.g., by genetic engineering techniques.

Sequence identity: The similarity between two nucleic acid sequences, ortwo amino acid sequences, is expressed in terms of the similaritybetween the sequences, otherwise referred to as sequence identity.Sequence identity is frequently measured in terms of percentage identity(or similarity or homology); the higher the percentage, the more similarthe two sequences are. Homologs or orthologs of human MATER protein, andthe corresponding cDNA or gene sequence, will possess a relatively highdegree of sequence identity when aligned using standard methods. Thishomology will be more significant when the orthologous proteins or genesor cDNAs are derived from species that are more closely related (e.g.human and chimpanzee sequences), compared to species more distantlyrelated (e.g., human and C. elegans sequences).

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smith &Waterman Adv. Appl. Math. 2: 482, 1981; Needleman & Wunsch J. Mol. Biol.48: 443, 1970; Pearson & Lipman Proc. Natl. Acad. Sci. USA 85: 2444,1988; Higgins & Sharp Gene, 73: 237–244, 1988; Higgins & Sharp CABIOS 5:151–153, 1989; Corpet et al. Nuc. Acids Res. 16, 10881–90, 1988; Huanget al. Computer Appls. in the Biosciences 8, 155–65, 1992; and Pearsonet al Meth. Mol. Bio. 24, 307–31, 1994. Altschul et al. (J. Mol. Biol.215:403–410, 1990), presents a detailed consideration of sequencealignment methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al. J.Mol. Biol. 215:403–410, 1990) is available from several sources,including the National Center for Biotechnology Information (NCBI,Bethesda, Md.) and on the Internet, for use in connection with thesequence analysis programs blastp, blastn, blastx, tblastn and tblastx.By way of example, for comparisons of amino acid sequences of greaterthan about 30 amino acids, the Blast 2 sequences function is employedusing the default BLOSUM62 matrix set to default parameters, (gapexistence cost of 11, and a per residue gap cost of 1). When aligningshort peptides (fewer than around 30 amino acids), the alignment shouldbe performed using the Blast 2 sequences function, employing the PAM30matrix set to default parameters (open gap 9, extension gap 1penalties).

An alternative indication that two nucleic acid molecules are closelyrelated is that the two molecules hybridize to each other understringent conditions. Stringent conditions are sequence-dependent andare different under different environmental parameters. Generally,stringent conditions are selected to be about 5° C. to 20° C. lower thanthe thermal melting point (T_(m)) for the specific sequence at a definedionic strength and pH. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of the target sequence remains hybridizedto a perfectly matched probe or complementary strand. Conditions fornucleic acid hybridization and calculation of stringencies can be foundin Sambrook et al. (In Molecular Cloning: A Laboratory Manual, CSHL, NewYork, 1989) and Tijssen (Laboratory Techniques in Biochemistry andMolecular Biology—Hybridization with Nucleic Acid Probes Part I, Chapter2, Elsevier, New York, 1993). Nucleic acid molecules that hybridizeunder stringent conditions to a human MATER protein-encoding sequencewill typically hybridize to a probe based on either an entire humanMATER protein-encoding sequence or selected portions of the encodingsequence under wash conditions of 2×SSC at 50° C.

Nucleic acid sequences that do not show a high degree of identity maynevertheless encode similar amino acid sequences, due to the degeneracyof the genetic code. It is understood that changes in nucleic acidsequence can be made using this degeneracy to produce multiple nucleicacid molecules that all encode substantially the same protein.

Specific binding agent: An agent that binds substantially only to adefined target. Thus a protein-specific binding agent bindssubstantially only the specified protein. By way of example, as usedherein, the term “MATER-protein specific binding agent” includesanti-MATER protein antibodies (and functional fragments thereof) andother agents (such as soluble receptors) that bind substantially only tothe MATER protein.

Anti-MATER protein antibodies may be produced using standard proceduresdescribed in a number of texts, including Harlow and Lane (Antibodies, ALaboratory Manual, CSHL, New York, 1988). The determination that aparticular agent binds substantially only to the specified protein mayreadily be made by using or adapting routine procedures. One suitable invitro assay makes use of the Western blotting procedure (described inmany standard texts, including Harlow and Lane (Antibodies, A LaboratoryManual, CSHL, New York, 1988)). Western blotting may be used todetermine that a given protein binding agent, such as an anti-MATERprotein monoclonal antibody, binds substantially only to the MATERprotein.

Shorter fragments of antibodies can also serve as specific bindingagents. For instance, Fabs, Fvs, and single-chain Fvs (SCFvs) that bindto a specified protein would be specific binding agents. These antibodyfragments are defined as follows: (1) Fab, the fragment which contains amonovalent antigen-binding fragment of an antibody molecule produced bydigestion of whole antibody with the enzyme papain to yield an intactlight chain and a portion of one heavy chain; (2) Fab′, the fragment ofan antibody molecule obtained by treating whole antibody with pepsin,followed by reduction, to yield an intact light chain and a portion ofthe heavy chain; two Fab′ fragments are obtained per antibody molecule;(3) (Fab′)2, the fragment of the antibody obtained by treating wholeantibody with the enzyme pepsin without subsequent reduction; (4)F(ab′)2, a dimer of two Fab′ fragments held together by two disulfidebonds; (5) Fv, a genetically engineered fragment containing the variableregion of the light chain and the variable region of the heavy chainexpressed as two chains; and (6) single chain antibody (“SCA”), agenetically engineered molecule containing the variable region of thelight chain, the variable region of the heavy chain, linked by asuitable polypeptide linker as a genetically fused single chainmolecule. Methods of making these fragments are routine.

Subject: Living multi-cellular vertebrate organisms, a category thatincludes both human and non-human mammals.

Target sequence: “Target sequence” is a portion of ssDNA, dsDNA or RNAthat, upon hybridization to a therapeutically effective oligonucleotideor oligonucleotide analog, results in the inhibition of expression. Forexample, hybridization of therapeutically effectively oligonucleotide toa Mater target sequence results in inhibition of MATER expression.Either an antisense or a sense molecule can be used to target a portionof dsDNA, since both will interfere with the expression of that portionof the dsDNA. The antisense molecule can bind to the plus strand, andthe sense molecule can bind to the minus strand. Thus, target sequencescan be ssDNA, dsDNA, and RNA.

Transformed: A transformed cell is a cell into which has been introduceda nucleic acid molecule by molecular biology techniques. As used herein,the term transformation encompasses all techniques by which a nucleicacid molecule might be introduced into such a cell, includingtransfection with viral vectors, transformation with plasmid vectors,and introduction of naked DNA by electroporation, lipofection, andparticle gun acceleration.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. A vector may include nucleic acidsequences that permit it to replicate in a host cell, such as an originof replication. A vector may also include one or more selectable markergenes and other genetic elements known in the art.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The singular terms “a,” “an,”and “the” include plural referents unless context clearly indicatesotherwise. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including explanations ofterms, will control. In addition, the materials, methods, and examplesare illustrative only and not intended to be limiting.

III. Human MATER Protein and Nucleic Acid Sequences

Embodiments provide MATER proteins and Mater nucleic acid molecules,including cDNA sequences. The prototypical Mater sequences are themurine and human sequences, and the use of these sequences to producetransgenic animals having increased or decreased levels of MATER proteinis provided, as are diagnostic methods to detect defects or alterationsin Mater expression or MATER protein production.

The full-length cDNA for human Mater (SEQ ID NO: 23) is determined to be3900 base pairs long (which is somewhat longer the mouse Mater cDNA,GenBank Accession # NM_(—)011860.1, SEQ ID NO: 5) and its ORF encodes aprotein of 1200 amino acids, having a predicted molecular weight ofapproximately 135.2 kDa and a predicted pI of about 6.08. The humanMater cDNA comprises the sequences shown in SEQ ID NO: 1 and SEQ ID NO:3, which are thought to be aligned approximately with the seventh andeleventh through fifteenth exons of the mouse Mater sequence,respectively. The human Mater mRNA is expressed in oocytes, as shown byin situ hybridization experiments (Example 3).

Human MATER protein (SEQ ID NO: 24) has been specifically localized byimmunofluorescence to human oocytes (Example 4). The ATG initiationcodon of the Mater cDNA lies within the context of the ANNATG motifassociated with vertebrate initiator codon (Kozak, 1991, J. Biol. Chem.266:19867). The human MATER protein comprises the deduced proteinsequences (SEQ ID NOs: 2 and 4) corresponding to the herein describedhuman Mater cDNA sequences (SEQ ID NOs: 1 and 3, respectively). Theseprotein fragments show low but significant sequence homology with theOPI/MATER protein from mouse (GenBank # NP_(—)035990, SEQ ID NO: 6). Inparticular, SEQ ID NO: 2 is 54% identical to residues 257–638 of murineMATER (SEQ ID NO: 6), while SEQ ID NO: 4 is 64% identical to residues854–1111 of murine MATER. The overall human MATER protein sequence (SEQID NO: 24) shares approximately 50% similarity with the murine MATERprotein sequence (SEQ ID NO: 6); the relevant sequence alignment isshown in FIG. 9. The depicted comparison between the human and murineMATER proteins was conducted using the BLAST 2 Sequences Program withparameter conditions of Expect 10 and Filter closed (NIH-NCBI).

Certain regions of the human Mater cDNA have been identified through thesequencing of human HTGSs, though no function or identity had beenpreviously assigned to those sequences. See, for instance, GenBankaccession numbers: AC024580 (GI=7705010, published May 4, 2000);AC012107 (GI=6088020, published Oct. 20, 1999, and updated asGI=7329252, published Mar. 28, 2000); and AC023887 (GI=7631054,published Apr. 21, 2000). These fragmentary and overlapping humansequences are up to 207 nucleotides in length and are scatteredthroughout the murine Mater cDNA sequence. The human ESTs individuallyshare up to 89% identity with the murine Mater cDNA over short,discontinuous regions. Oligonucleotides according to the currentdisclosure may be chosen to avoid these ESTs. Overall, the first 75nucleotides of human Mater (1–75) and the corresponding deduced aminoacids (1–25) were determined by comparison to published human genomicDNA sequences, while the remainder of the Mater sequence was determinedby direct cloning and sequencing of human ovarian cDNAs.

Mater is a single-copy maternal effect gene, the protein product ofwhich is required for early embryonic survival. Although eggs lackingMATER protein can be fertilized, morphologic signs of deterioration areobserved as early as the 2-cell stage beyond which the mutant embryos donot progress. The human MATER protein with 1200 amino acids is predictedto have similar molecular domains to those seen in the murine MATERprotein (1111 amino acids). As described by Tong et al. (Mamm. Genome11:281–287, 2000), the mouse MATER protein contains a five-foldhydrophilic repeat (26–27 amino acid) near its N-terminus, a shortleucine-zipper and a fourteen-fold leucine-rich repeat (28–29 aminoacid) near its carboxyl terminus (based on a comparison to the murineMATER protein, as described in Tong et al., Mamm. Genome 11, 281–287,2000). The hydrophilic repeat has low homology with a cytoskeletonalprotein (neurofilament), raising the possibility that this regionmediates interactions that anchor MATER in the cytoplasm. The presenceof the leucine-rich domain, as well as a short leucine zipper, bothmotifs known to mediate protein-protein interactions (Kajava, J. Mol.Biol. 277, 519–527, 1998; Buchanan & Gay, Prog. Biophys. Mol. Biol. 65,1–44, 1996), suggests that MATER may affect embryonic progressionthrough intermediate factor(s), one or more of which binds directly toMATER in the cytoplasm.

Using antisera against a mouse MATER peptide (residues 1093 through 1111of murine MATER, SEQ ID NO: 6), the inventors have demonstrated thathuman MATER protein is present in oocytes in human ovary sections(Example 4). Likewise, using mouse-derived nucleotide sense andantisense probes for in situ hybridization experiments, it has beendemonstrated that human oocytes express Mater mRNA (Example 3).

The inventors have further characterized murine MATER as to its functionin mammalian embryogenesis. This protein is a “maternal” protein,generated in the oocyte prior to fertilization, and therefore encodedfor by a maternal gene. MATER is unusual in that it persists (asmeasured by protein level) into the late blastocyst stage of embryonicdevelopment. Functional MATER is required for female fertility; zygotesthat arise from Mater null oocytes do not progress beyond the two-cellstage; this is true regardless of what Mater genotype male produced thesperm. The protein is cytoplasmic, with definite exclusion from thenucleus.

The invention is illustrated by the following non-limiting Examples.

EXAMPLE 1 Characterization of the Murine MATER Protein

This example provides several methods for examining MATER proteins andnucleic acids in a mammalian system; the murine system is used.

Methods:

Isolation of oocytes, eggs and embryos. All experiments were conductedin compliance with the guidelines of the Animal Care and Use Committeeunder an approved animal study protocol. Resting, growing and fullygrown oocytes were dissected from 1, 10 and 21 day old (d/o) mouseovaries, respectively, and eggs were isolated from gonadotrophinstimulated female mice (Tong et al., J. Biol. Chem. 270, 849–853, 1995;Rankin et al., Development 125, 2415–2424, 1998). Embryos were obtainedby mating gonadotrophin stimulated females with males and 1-cellzygotes, 2-cell embryos, morulae and blastocysts were flushed from theoviducts at 1, 2, 3 and 4 days, respectively, counting the morning afterhCG administration as day 1. Embryos were either incubated in M16 media(37° C., 5% CO₂), fixed in 1% paraformaldehyde for subsequent confocalmicroscopy, or frozen at −80° C. for RNA and protein analyses. Ovarieswere fixed (10% formalin) and embedded in paraffin prior to sectioningand staining with hematoxylin and eosin (American Histolabs,Gaithersburg, Md.).

Detection of transcripts and proteins. Mater, ZP3, β-actin, cyclophilinand 28S-rRNA transcripts were detected by RNase protection assays aspreviously described (Tong & Nelson, Endocrinology 140, 3720–3726, 1999;Tong et al., J. Biol. Chem. 270, 849–853, 1995) using an RPA II kit(Ambion, Austin, Tex.).

Rabbits were immunized with a KLH conjugated MATER peptide (amino acids1093–1111) to obtain a monospecific antisera. After incubatingimmunoblots with MATER antisera (1:1000, two hours, 20° C.), antibodybinding was detected by ECL using a HRP-conjugated goat anti-rabbitantibody (Amersham Pharmacia Biotech, Piscataway, N.J.). Fixed oocytes,eggs or embryos were incubated with MATER antisera (1:8000, 4° C., 16hours) and imaged by laser-scanning confocal microscopy (LSM 5; Zeiss,Thornwood, N.Y.) using Cy⁵-conjugated goat anti-rabbit IgG (1:200).

Protein synthesis and immunoblotting. Embryos were incubated in 100 μlof M16 medium containing L-³⁵S-methionine (0.5 Ci/ml, Amersham) at 37°C. for 4 hours. At the end of radiolabeling, 1-cell and 2-cell embryoswere harvested at 26 hours and 48 hours post-hCG/mating, respectively.After washing with 1% BSA in 10 mM Tris-HCl (pH 7.4), 1 mM EDTA and 140mM NaCl, ten embryos were dissolved directly in 10 μl of sample buffer(1) directly or (2) after treatment with 2% Triton X-100/0.3 M KCl toextract the TRC and p35 (Conover et al., Dev. Biol. 144, 392–404, 1991).After SDS-PAGE and fluorography as previously reported (Tong et al., J.Biol. Chem. 270, 849–853, 1995), radioactive incorporation wasdetermined by a phosphoimager and ImageQuant software (MolecularDynamics, Amersham Pharmacia Biotech, Piscataway, N.J.).

Production of Mater null mice. Constructs use to produce Mater null miceare shown in FIG. 2. To construct a targeting vector in pPNT (Tybulewiczet al., Cell 65, 1153–1163, 1991), a 1.5 kbp SacI-EcoRI DNA fragmentcontaining the first two exons of murine Mater (Tong et al., Mamm.Genome 11, 281–287, 2000) was inserted between the PGK-Neo and PGK-TKcassettes and a 2 kbp EcoRI-BamHI fragment containing exons 3 and 4 wasinserted upstream of the PCK-Neo. After linearization andelectroporation into embryonic stem (ES) cells (Redmond et al., Nat.Genet. 20, 344–351, 1998), the presence of the mutant allele inchemically selected clones was detected as 3.5 kbp and 4.0 kbp XbaIfragments with 5′ and 3′ (or Neo) probes, respectively. Both the 5′ and3′ probes detected the normal allele as a 9.4 kbp XbaI fragment. C57BL/6blastocysts were injected with 8–12 ES cells derived from fiveindependently selected clones. Two cell lines that gave rise tocoat-color chimeric animals were mated with C57BL/6 females andtransmitted the Mater mutation through the germ line. The absence ofMater transcripts in homozygous null females was confirmed by northernblot analysis as described previously (Tong & Nelson, Endocrinology 140,3720–3726, 1999).

Reproductive performance. Vaginal smears were obtained daily to examinefour or more estrus cycles of 6–10 week-old female mice (Rugh, TheMouse: Its Reproduction and Development 210 Oxford University Press,Oxford, 1991). Mating behavior was evaluated by the presence of avaginal plug on the morning after mating with fertile males.

Zygotic gene transcription assays. A 5–10 ρl aliquot of 100 mM BrUTP,140 mM KCl and 2 mM Pipes, pH 7.4 was microinjected into the cytoplasmof embryos isolated 28 hours (1-cell embryos) and 48 hours (2-cellembryos) following hCG and mating, essentially as previously described(Bouniol et al., Exp. Cell Res. 218, 57–62, 1995). After incubation inM16 medium in 5% CO₂ for one hour at 37° C., the injected embryos werefixed in 1% paraformaldehyde.

BrUTP incorporation into RNA was assayed with a mouse anti-BrdUmonoclonal antibody (Sigma, 1:1000, 16 hours, 4° C.). After washing inPBS containing 3% BSA, a FITC-conjugated goat anti-mouse IgG antibodywas used to image the embryos by confocal microscopy; fluorescence wasrecorded in arbitrary units. This assay primarily reflects de novo RNApolymerase II activity, because poor penetration of the monoclonalantibody into nucleoli prevents measurement of BrUTP incorporation intoribosomal RNA (Wansink, et al., J. Cell Biol. 122, 283–293, 1993).

Results

Mater Transcript and Protein Expression

Mater mRNA was first detected as oocytes entered into their growth phase(30–50 μm) (FIG. 1A). Mater transcripts were most abundant infully-grown oocytes (75–85 μm) and, like many oocyte transcripts(Bachvarova, in Developmental Biology: Oogenesis 1, 453–524, PlenumPress, New York, 1985; Epifano et al., Development 121, 1947–1956,1995), were degraded during meiotic maturation and subsequent ovulation.Mater mRNA was not detected in the early embryo (FIG. 1A), although itspresence in EST databases derived from early embryos suggest that sometranscripts may have escaped destruction.

The initial accumulation of MATER protein during oogenesis was similarto that observed for Mater transcripts (compare FIG. 1A with FIG. 1B).However, unlike the virtual disappearance of the transcripts, the MATERprotein persisted during preimplantation development until the lateblastocyst stage (FIG. 1B). Using confocal microscopy, MATER protein waslocated in the cytoplasm of growing oocytes and noticeably excluded fromthe nucleus. MATER was more concentrated in the cortical region ofoocytes and early embryos (one- and two-cell stages), and thisperipheral pattern persisted later in preimplantation development with ahigher concentration of MATER in the outer trophectodermal cellscompared to the inner cell mass of blastocysts (FIG. 1C).

Mater Null Animals

To determine the function of MATER protein, Mater null mouse lines weregenerated, in which homozygous females did not express either Matertranscripts or protein. Mater null mice were born in the expectedMendelian ratios with equal sex distribution. No phenotypicabnormalities were observed from birth through adulthood.

Ovaries and uteri from Mater null mice appeared morphologically normal(FIG. 6). Mater null ovaries had a normal complement of primordialfollicles and all stages of follicular development were normallyrepresented; corpora lutea, indicating past spontaneous ovulation, werealso present (FIG. 5).

Ova lacking Mater were fertilized normally in vivo (RIG. 1). The numberand morphology of zygotes and 2-cell embryos from the Mater null femaleswere similar to those from normal females (FIG. 5). However, by 3 or 4days after mating the embryos from Mater null females still remained atthe 2-cell stage or had begun to degenerate (compare FIGS. 3D and 3Gwith 3E and 3H). Thus, fertilization is normal in Mater null females andthe resulting zygotes can progress through the first cleavage, butsubsequent development is arrested at the 2-cell stage. This indicatesthat an arrest of early embryogenesis accounts for the infertility inMater null females.

Two mouse lines with a Mater null mutation (T54 and T85) were generated(FIGS. 2A and 2B) and neither Mater transcript (FIG. 2C) nor protein(FIG. 2D) were detected in homozygous null females.

Reproductive Phenotype of Mater Null Animals

Matings of heterozygous Mater null parents produced average sizedlitters (8.5±1.9) with normal Mendelian and equal sex distribution ofthe null allele in progeny. Homozygous null animals appeared normal andmales were as fertile as normal littermates. However, homozygous nullfemales produced no litters, even after five months of mating (Table 1)which implicated Mater as a maternal effect gene.

TABLE 1 Fertility and Mendelian allele ratio of mice with Mater mutationMating pairs Litter sizes Mendelian ratios ♂ × ♀ n* mean ± s.e.m. +/++/− −/− Mater^(+/+) × Mater^(+/+) 11 7.5 ± 1.8 1.00 0   0   Mater^(+/+)× Mater^(+/−) 10 7.4 ± 1.9 n.d. ^(†) n.d. n.d. Mater^(+/+) × Mater^(−/−)9 0 n.a. ^(‡) n.a. n.a. Mater^(−/−) × Mater^(+/+) 8 7.6 ± 2.4 0   1.000   Mater^(−/−) × Mater^(+/−) 18 8.4 ± 1.9 0   0.47 0.53 Mater^(+/−) ×Mater^(+/−) 15 8.5 ± 1.9 0.21 0.57 0.22 *n, the number of mating pairs,^(†) n.d., not done, ^(‡) n.a., not applicable.

Sexually mature Mater null females exhibited regular 5.67±0.22 daysestrus cycles, similar to those observed in normal females (5.40±0.23days). Mating occurred normally, as demonstrated by the appearance ofvaginal plugs in about 70% of either Mater null or normal females, afterexposure to fertile males. The Mater null ovary had a normal complementof primordial follicles, with all stages of follicular development aswell as corpora lutea indicating past spontaneous ovulations (FIGS. 5Aand 5B). In addition, the Mater null females ovulated normally afterstimulation with exogenous gonadotropins. However, homozygous nullfemales produced no litters even after five months of mating with normalmales, while the homozygous null males and heterozygous females hadnormal fertility.

The mean number (±s.e.m.) of ovulated eggs recovered from null(27.6±4.5, n=14) was not statistically different (p>0.05) from thatobtained from normal (27.7±4.3, n=12) females (FIG. 5D).Morphologically, the two groups of eggs were indistinguishable from oneanother (FIG. 5C).

Fertilization and Embryo Development in Mater Null Animals

To evaluate in vivo fertilization and early development, embryos wereisolated from normal and Mater null females at 1, 2, 3 and 4 days aftermating with normal males. Fertilization occurred in eggs lacking MATER,as demonstrated by the presence of two pronuclei (arrows, FIGS. 3A and3B). The number and morphology of 1-cell zygotes and 2-cell embryosobtained in vivo from the Mater null females were similar to thoseobserved from normal females, although the 2-cell mutant embryosappeared less healthy, displaying cytoplasmic granulations (FIGS.3A–3F). Embryos obtained from the Mater null female mice 3 or 4 daysafter mating still remained at the 2-cell stage (or had begun todegenerate), while embryos from normal mice had progressed to the morulaor blastocyst stages, respectively

An equal percentage (˜70%) of fertilized 1-cell zygotes derived fromMater null and normal females progressed in vitro to the 2-cell stage.However, after four days in culture, embryos lacking MATER remainedarrested at the 2-cell stage (most were degenerating) (FIG. 3K); embryosfrom the normal females developed to the blastocyst stage in the sametime (FIG. 3J). Thus, although fertilization appears normal and zygoteswithout MATER can progress through the first cleavage, subsequentembryonic development is arrested at the 2-cell stage.

Characterization of Transcription in Mater Null Animals

The early arrest phenotype seen in Mater null mice is reminiscent of the2-cell block that occurs after exposure of mouse embryos to α-amanitin(Schultz, Bioessays 15:531–538, 1993; Flach et al., EMBO J. 1, 681–686,1982), a mushroom toxin that binds to RNA polymerase II and preventselongation of transcription beyond dinucleotides (Vaisius & Wieland,Biochemistry 21, 3097–3101, 1982). Normal embryonic transcription isfirst detected late in the 1-cell zygote at a level that is ˜20% that ofthe 2-cell embryo (Aoki et al., Dev. Biol. 181, 296–307, 1997), butthese nascent transcripts are poorly, if at all, translated into protein(Nothias et al., EMBO J. 15, 5 715–5725, 1996).

To assess de novo RNA polymerase II activity of the early Mater nullembryo, bromo-UTP (BrUTP) incorporation was assayed with a monoclonalantibody specific to BrUTP using laser-scanning confocal microscopy,essentially as described previously (Bouniol et al, Exp. Cell Res. 218,57–62, 1995) (FIGS. 4A–4G). Morphologically there was a dramaticdifference in the amount of BrUTP detected in the nuclei of normalembryos 28 hours (FIG. 4B) and 48 hours (FIG. 4D) after mating comparedto those lacking MATER (FIGS. 4A and 4C). BrUTP incorporation in embryoswithout MATER was marginally greater than that of uninjected controls(FIGS. 4E and 4F) and de novo transcription was less than 5% thatobserved in normal 2-cell embryos (FIG. 4G).

Characterization of TRC in Mater Null Animals

During meiotic maturation and ovulation, more than 50% of maternal RNAis lost in normal mice (Bachvarova, in Developmental Biology: Oogenesis1, 453–524, Plenum Press, New York, 1985). This degradative process alsooccurs in females lacking MATER with loss of ribosomal RNA (FIG. 4H) andat least some mRNAs (β-actin, ZP3, cyclophilin, and GAPDH). Normally,the subsequent activation of the embryonic genome at the 2-cell stage ispreceded by the transient transcription and translation of a subset ofgene products, some of which form the distinctive Transcription RelatedComplex (TRC) (Bolton et al., J. Embryol. Exp. Morphol. 79:139–163,1984; Nothias et al., EMBO J. 15:5715–5725, 1996; Conover et al., Dev.Biol. 144:392404, 1991). This complex, first observed in the early2-cell embryo, is not formed in the presence of α-amanitin.

To assay for the TRC, 1- and 2-cell embryos derived from Mater nullfemales mated with normal males were incubated with ³⁵S-methionine. Noglobal differences in the amount of ³⁵S-methionine incorporation or inprotein profiles were noted in comparing normal 1-cell zygotes and thoselacking MATER (FIG. 4I, left panel). Although TRC complexes weredetected in embryos without MATER at the 2-cell stage (FIG. 4I, middleand right panels), they appeared somewhat less abundant (˜60% ofnormal), which could reflect abnormal protein synthesis in the embryoslacking MATER This indicates that MATER is not absolutely critical forinitiation of all transcription-translation machinery in early embryos.

Although this striking decrease in transcription could reflectgeneralized morbidity of the embryo, a similar decrease in 1-cellzygotes, which appear quite healthy, as well as absence of an equallydramatic decrease in de novo protein synthesis (FIG. 4I) suggest thatthe depressed levels of transcription are due either directly, orindirectly to the absence of MATER.

Proposed Function(s) of Mammalian MATER

The homology of the leucine-rich repeat domain of mammalian MATERprotein with porcine ribonuclease inhibitor implies that MATER may actas an inhibitor of cytoplasmic RNase. If this is true, RNA in embryoslacking MATER might be subject to extensive degradation, perhaps anaccentuation of the normal turnover of RNA that occurs during thematernal-to-zygotic transition in the early embryo. The degradation ofRNAs, including those that encode proteins required for transcription,could account for the phenotype observed in Mater null embryos. Thisseems unlikely; however, because in the absence of Mater mutant embryosincorporate ³⁵S-methionine into proteins and no major difference inprotein profiles are observed in comparison with normal embryos. Theseresults indicate that sufficient mRNA, tRNA and ribosomal RNA arepresent in the mutant embryos to support protein biosynthesis, althoughit does not preclude the degradation of a targeted subset of RNArequired for early embryonic survival.

Transcription-dependent protein synthesis occurs in two phases in the2-cell embryo, an early minor activation (2–4 hours after the firstmitotic division) that is characterized by appearance of the TRCproteins, and a later major activation that occurs in G₂ (8–10 hoursafter the first mitotic division). The later activation initiatesdevelopmental programs required for progression beyond the 2-cell stage(Schultz, Bioessays 15, 531–538, 1993). The observed decrease inembryonic transcription in embryos lacking MATER could result fromdeterioration of transcription machinery, decreased access to genomicDNA or abnormalities in the chromatin template within the nucleus.However, the presence of the TRC proteins in Mater null animalsindicates that MATER is not critical for the early phase oftranscription-dependent protein synthesis in 2-cell embryos. It furthersuggests that the transcription machinery, and its ability to accessnuclear DNA, is largely intact in Mater null embryos (as it appears tobe in Mater null oocytes, which are also transcriptionally active).

The TRC proteins resulting from the early burst of transcription areassociated with the nucleus (Schultz, Bioessays 15, 531–538, 1993).These proteins could relieve the late transcriptional repressionrequired for progression beyond the 2-cell stage via mechanismsinvolving MATER. Additionally, it has been reported that mouse oocytesand 1-cell embryos lack coactivator(s) required for enhancer-dependenttranscription in 2-cell embryos (Lawinger et al., J. Biol. Chem. 274,8002–8011, 1999). The nature of these coactivators is unknown; they mayarise by modification of pre-existing maternal proteins or from earlytranscription-dependent protein synthesis in the 2-cell embryo. Suchprocess may involve cytoplasmic MATER Alternatively, MATER might beinvolved in one or more interactions that are required for cell cycleprogression beyond the 2-cell stage. Identification and characterizationof proteins that interact with MATER will provide insights into the roleof this maternal protein in promoting embryonic survival and earlydevelopment.

Mater is the first maternal effect gene demonstrated to play a criticalrole in early mammalian development and the observed sterile phenotyperaises the possibility that the absence of a similar molecule could be acause of human infertility.

EXAMPLE 2 Identification of the Human Mater cDNA

The human Mater cDNA was identified based on its homology to the mouseMater cDNA sequence by searching the collection of human High ThroughputGenomic (HTG) Sequences, maintained by NCBI and the National Library ofMedicine. Using the isolated and discontinuous nucleotide fragmentsidentified in this search, the inventors devised oligonucleotide primerspairs (for instance, SEQ ID NOs: 7 and 8, 19 and 20, and 21 and 22) thatwhere used to amplify Mater sequence from purified human ovarian DNA ormRNA/cDNA, and to isolate a partial cDNA clone from a human cDNAlibrary, using standard techniques.

The sequence of two long fragments of the human Mater cDNA is shown inSEQ ID NO: 1 and 3. Together, these two long cDNA fragments constitute2213 nucleotides of the complete human Mater cDNA.

The deduced human MATER protein portions encoded cDNA fragment 1 (SEQ IDNO: 1) and fragment 2 (SEQ ID NO: 3) are shown in SEQ ID NOs: 2 and 4,respectively.

Using these sequences, the remainder of the human Mater cDNA has beenidentified; the sequence of the full length cDNA is shown in SEQ ID NO:23. Overall, the first 75 nucleotides of human Mater (1–75) and thecorresponding deduced amino acids (1–25) were determined by comparisonto published human genomic DNA sequences, while the remainder of theMater sequence was determined by direct cloning and sequencing of humanovarian cDNAs. The complete sequence of the human MATER protein is shownin SEQ ID NO: 24.

EXAMPLE 3 Localization of Human Mater Transcript

This example provides one method for detecting expression of the humanMater transcript.

To produce in situ hybridization probes, human genomic DNA was amplifiedusing the following primers:

5′-primer: 5′-TTTCACATGAACATCCTTCTCC-3′; (SEQ ID NO: 7) 3′-primer:5′-AGTGCTGGAGGCAGAAGGAAG-3′. (SEQ ID NO: 8)The resultant amplified nucleic acid molecule product size was 496 bp,and is predicted to fall within exon 7 of the human Mater gene, based oncomparison to the murine Mater exon-intron map. This fragment wassubcloned into pBluscript vector as a DNA template. Both ³⁵S-UTP-labeledsense and antisense sequences were synthesized by in vitro transcriptionusing T3 and T7 RNA polymerases.

In situ hybridization was carried out essentially as describedpreviously (Tong & Nelson, Endocrinology 140:3720–3726, 1999). Briefly,the probes were prepared by alkaline hydrolysis and hybridized withfrozen human ovarian sections at 60° C. for 24 hours. After dipping inKodak NTB-2 emulsion, the slides were exposed to film for 2–3 days andthe autoradiographs developed.

The frozen human ovarian sections were hybridized with the radio-labeledsense (FIGS. 7A and 7C) and antisense (B and D) probes. The slides werestained with hematoxylin and eosin. For each probe, bright-field (A andB) and dark-field (C and D) images are displayed. Mater transcript waslocalized to oocytes.

EXAMPLE 4 Localization of Human MATER Protein

This example provides one method for examining the localization of humanMATER protein.

In situ localization was carried out essentially as described for murinesamples (see, Tong and Nelson, Endocrinology 140:3720–3726, 1999).Briefly, rabbit polyclonal antibody to a murine MATER C-terminal peptide(residues 1093 through 1111 of SEQ ID NO: 6) was prepared as describedin Example 9C. Frozen human ovarian sections were incubated with thisantiserum (1:200), and FITC-conjugated goat anti-rabbit IgG antiserumwas used as the secondary antibody to detect human MATER protein in theoocyte (FIG. 8A). FIG. 8B shows the phase contrast images correspondingto the samples in FIG. 8A.

EXAMPLE 5 Methods of Making Human Mater cDNA

The original means by which the Mater cDNA was identified and obtainedis described above. With the provision of the sequence of the largeportions of the MATER protein (SEQ ID NOs: 2 and 4) and encoding nucleicacid molecules (SEQ ID NOs: 1 and 3), nucleotide amplification (such aspolymerase chain reaction (PCR)) now may be utilized in a simple methodfor producing the Mater cDNA.

Total RNA is extracted from human cells by any one of a variety ofmethods well known to those of ordinary skill in the art. Sambrook etal. (In Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989)and Ausubel et al. (In Current Protocols in Molecular Biology, GreenePubl. Assoc. and Wiley-Intersciences, 1992) provide descriptions ofmethods for RNA isolation. Because MATER is expressed in oocytes, humancell lines derived from oocytes or ovaries can be used as a source ofsuch RNA. The extracted RNA is then used as a template for performingreverse transcription-polymerase chain reaction (RT-PCR) amplificationof cDNA. Methods and conditions for RT-PCR are described in Kawasaki etal, (In PCR Protocols, A Guide to Methods and Applications, Innis et al.(eds.), 21–27, Academic Press, Inc., San Diego, Calif., 1990).

The selection of amplification primers will be made according to theportion(s) of the cDNA that is to be amplified. Primers may be chosen toamplify a segment of a cDNA or the entire cDNA molecule. Variations inamplification conditions may be required to accommodate primers andamplicons of differing lengths and composition; such considerations arewell known in the art and are discussed for instance in Innis et al.(PCR Protocols, A Guide to Methods and Applications, Academic Press,Inc., San Diego, Calif., 1990). By way of example, the majority of thecoding portion of the human MATER cDNA molecule (approximately 2.8 kb)may be amplified using the following combination of primers:

5′-primer (part of predicted exon 7): 5′-CAGGAATTGGGAAATCGGCTCTCTAG-3′;(SEQ ID NO.: 9) 3′-primer (end of predicted exon 15):5′-CCAAATGCTTTGTGTTTATTTAATTCC-3′. (SEQ ID NO.: 10)One of ordinary skill can use the full length human Mater cDNA sequenceprovided herein (SEQ ID NO: 23) to devise primers that could be used toamplify the entire cDNA using known techniques.

The following set of four primers can be used to amplify ˜950 bp at the5′-end of human Mater cDNA:

The 5′-primers are the universal adapter primers (ADP) for 5′-RACE-PCRas follows (OriGene Technologies, Inc, Rockville, Md.):

ADP1: 5′-CGGAATTCGTCACTCAGCG-3′ (SEQ ID NO.: 11) ADP2:5′-AGCGCGTGAATCAGATCG-3′ (SEQ ID NO.: 12)The 3′-primers are gene-specific primers (GSP) for human MATER cDNAwithin exon 7, as follows:

GSP1: 5′-ATTCCCTGGTAGAGTCCACCTTGC-3′ (SEQ ID NO.: 13) GSP2:5′-ACAGCACGATCCTTCTGGCTAGAG-3′. (SEQ ID NO.: 14)GSP1 is used as a primer for reverse transcription of human ovarian RNAto synthesize cDNA. The reverse transcribed cDNAs are then adapted atthe 5′-end by ADP1 primer, then amplified for the first round of PCRusing ADP1 and GSP1 primers. The second round of PCR amplification isfollowed using PCR products from the first round as template, and ADP2and GSP2 as primers.

Fragment 1 of the human Mater cDNA (SEQ ID NO: 1) may be amplified usingthe following combination of primers:

5′-primer: 5′-CAAGCTCCGGTGACGGAGATCAT-3′; (SEQ ID NO.: 15) 3′-primer:5′-AGCTGGAGGCAGAAGGAAGATG-3′. (SEQ ID NO.: 16)

The 3′-terminal region of the human Mater cDNA molecule (also referredto herein as human Mater cDNA fragment 2, SEQ ID NO: 3) may be amplifiedusing the following combination of primers:

5′-primer: 5′-TCTGGCCTCAGCCCTCGTCAGCTTGAC-3′; (SEQ ID NO.: 17)3′-primer: 5′-CCAAATGCTTTGTGTTTATTTAATTCC-3′. (SEQ ID NO.: 18)

These primers are illustrative only; one skilled in the art willappreciate that many different primers may be derived from the providedcDNA sequence in order to amplify particular the indicated regions MatercDNA, as well as to complete the sequence of the human Mater cDNA.

Re-sequencing of PCR products obtained by these amplification proceduresis recommended; this will facilitate confirmation of the amplifiedsequence and will also provide information on natural variation on thissequence in different populations or species. Oligonucleotides derivedfrom the provided Mater sequences provided may be used in suchsequencing methods.

Orthologs of human Mater can be cloned in a similar manner, where thestarting material consists of cells taken from a non-human species.Orthologs will generally share at least 80% sequence homology with thedisclosed human Mater cDNA. Where the non-human species is more closelyrelated to humans, the sequence homology will in general be greater.Closely related orthologous Mater molecules may share at least 82%, atleast 85%, at least 90%, at least 91%, at least 93%, at least 95%, or atleast 98% sequence homology with the disclosed human sequences.

Oligonucleotides derived from the human Mater cDNA sequence (SEQ ID NO:23), or fragments thereof (such as SEQ ID NOs: 1 and 3), are encompassedwithin the scope of the present disclosure. Preferably, sucholigonucleotide primers will comprise a sequence of at least 23consecutive nucleotides of the Mater nucleic acid sequence. To enhanceamplification specificity, oligonucleotide primers comprising at least25, 30, 35, 40, 45 or 50 consecutive nucleotides of these sequences mayalso be used. These primers for instance may be obtained from any regionof the disclosed sequences. By way of example, the human Mater cDNA, ORFand gene sequences may be apportioned into about halves or quartersbased on sequence length, and the isolated nucleic acid molecules (e.g.,oligonucleotides) may be derived from the first or second halves of themolecules, or any of the four quarters. The murine Mater cDNA, shown inSEQ ID NO: 5, can be used to illustrate this. The human Mater cDNA is3447 nucleotides in length and so may be hypothetically divided intoabout halves (nucleotides 1–1723 and 1724–3447) or about quarters(nucleotides 1–862, 863–1723, 1724–2586 and 2587–3447). The human cDNAcan be likewise apportioned, or can be described in terms of thefragments presented herein (e.g., fragment 1, corresponding to aroundabout nucleotide 1700 through around about 1950 of murine Mater; andfragment 2 of the human Mater cDNA, corresponding to the 3′-terminalregion of murine Mater cDNA, around about nucleotide 2500 through thepolyA tail of the murine Mater cDNA).

Nucleic acid molecules may be selected that comprise at least 15, 20,23, 25, 30, 35, 40, 50 or 100 consecutive nucleotides of any of these orother portions of the human Mater cDNA. Thus, representative nucleicacid molecules might comprise at least 15 consecutive nucleotides of thehuman Mater cDNA (SEQ ID NO: 23), or fragment 1 or fragment 2 of thedisclosed human Mater coding sequence (SEQ ID NOs: 1 and 3,respectively).

EXAMPLE 6 Cloning of the Mater Genomic Sequence (or Gene)

The Mater cDNA sequence and fragments described above does not containintrons, upstream transcriptional promoter or regulatory regions ordownstream transcriptional regulatory regions of the Mater gene. It ispossible that some mutations in the Mater gene that may lead to defectsin embryo development, infertility, or reduced fertility are notincluded in the cDNA but rather are located in other regions of theMater gene. Mutations located outside of the open reading frame thatencodes the MATER protein are not likely to affect the functionalactivity of the protein, but rather are likely to result in alteredlevels of the protein in the cell. For example, mutations in thepromoter region of the Mater gene may prevent transcription of the geneand therefore lead to the complete absence of the MATER protein in thecell.

Additionally, mutations within intron sequences in the genomic gene mayalso prevent expression of the MATER protein. Following transcription ofa gene containing introns, the intron sequences are removed from the RNAmolecule in a process termed splicing prior to translation of the RNAmolecule which results in production of the encoded protein. When theRNA molecule is spliced to remove the introns, the cellular enzymes thatperform the splicing function recognize sequences around the intron/exonborder and in this manner recognize the appropriate splice sites. Ifthere is a mutation within the sequence of the intron close to thejunction of the intron with an exon, the enzymes may not recognize thejunction and may fail to remove the intron. If this occurs, the encodedprotein will likely be defective. Thus, mutations inside the intronsequences within the Mater gene (termed “splice site mutations”) mayalso lead to defects in embryo development However, knowledge of theexon structure and intronic splice site sequences of the Mater gene isrequired to define the molecular basis of these abnormalities. Theprovision herein of the Mater cDNA sequence enables the cloning of theentire Mater gene (including the promoter and other regulatory regionsand the intron sequences) and the determination of its nucleotidesequence. With this information in hand, diagnosis of a geneticpredisposition to fertility defects based on DNA analysis willcomprehend all possible mutagenic events at the Mater locus.

The Mater gene may be isolated by one or more routine procedures,including direct sequencing of one or more BAC or PAC clones thatcontain the Mater sequence.

With the sequences of human Mater cDNA and gene in hand, primers derivedfrom these sequences may be used in diagnostic tests (described below)to determine the presence of mutations in any part of the genomic Matergene of a patient. Such primers will be oligonucleotides comprising afragment of sequence from the Mater gene (intron sequence, exon sequenceor a sequence spanning an intron-exon boundary) and may include at least10 consecutive nucleotides of the Mater cDNA or gene. It will beappreciated that greater specificity may be achieved by using primers ofgreater lengths. Thus, in order to obtain enhanced specificity, theprimers used may comprise 15, 17, 20, 23, 25, 30, 40 or even 50consecutive nucleotides of the Mater cDNA or gene. Furthermore, with theprovision of the Mater intron sequence information the analysis of alarge and as yet untapped source of patient material for mutations willnow be possible using methods such as chemical cleavage of mismatches(Cotton et al., Proc. Natl. Acad. Sci. USA 85:4397–4401, 1985; Montandonet al., Nucleic Acids Res. 9:3347–3358, 1989) and single-strandconformational polymorphism analysis (Orita et al., Genomics 5:874–879,1989).

Additional experiments may be performed to identify and characterizeregulatory elements flanking the Mater gene. These regulatory elementsmay be characterized by standard techniques including deletion analyseswherein successive nucleotides of a putative regulatory region areremoved and the effect of the deletions are studied by either transientor long-term expression analyses experiments. The identification andcharacterization of regulatory elements flanking the genomic Mater genemay be made by functional experimentation (deletion analyses, etc.) inmammalian cells by either transient or long-term expression analyses.

It will be apparent to one skilled in the art that either the genomicclone or the cDNA or sequences derived from these clones may be utilizedin applications, including but not limited to, studies of the expressionof the Mater gene, studies of the function of the MATER protein, thegeneration of antibodies to the MATER protein diagnosis and therapy ofMATER deleted or mutated patients to prevent or treat the defects inembryo development. Descriptions of applications describing the use ofMater cDNA, or fragments thereof, are therefore intended to comprehendthe use of the genomic Mater gene.

It will also be apparent to one skilled in the art that homologs of thisgene may now be cloned from other species, such as the rat or a monkey,by standard cloning methods. Such homologs will be useful in theproduction of animal models of onset and development of autoimmuneinfertility, and early embryogenesis. In general, such orthologous Matermolecules will share at least 70% sequence identity with the human Maternucleic acid disclosed herein; more closely related orthologoussequences will share at least 75%, at least 80%, at least 90%, at least95%, or at least 98% sequence identity with this sequence.

EXAMPLE 7 Mater Nucleic Acid and Amino Acid Sequence Variants

With the provision of human MATER protein fragments and correspondingnucleic acid sequences herein, the creation of variants of thesesequences is now enabled.

Variant MATER proteins include proteins that differ in amino acidsequence from the human MATER sequences disclosed but that share atleast 65% amino acid sequence homology with the provided human MATERprotein. Other variants will share at least 70%, at least 75%, at least80%, at least 90%, at least 95%, or at least 98% amino acid sequencehomology. Manipulation of the nucleotide sequence of Mater usingstandard procedures, including for instance site-directed mutagenesis orPCR, can be used to produce such variants. The simplest modificationsinvolve the substitution of one or more amino acids for amino acidshaving similar biochemical properties. These so-called conservativesubstitutions are likely to have minimal impact on the activity of theresultant protein. Table 2 shows amino acids that may be substituted foran original amino acid in a protein, and which are regarded asconservative substitutions.

TABLE 2 Original Residue Conservative Substitutions Ala ser Arg lys Asngln; his Asp glu Cys ser Gln asn Glu asp Gly pro His asn; gln Ile leu;val Leu ile; val Lys arg; gln; glu Met leu; ile Phe met; leu; tyr Serthr Thr ser Trp tyr Tyr trp; phe Val ile; leu

More substantial changes in enzymatic function or other protein featuresmay be obtained by selecting amino acid substitutions that are lessconservative than those listed in Table 2. Such changes include changingresidues that differ more significantly in their effect on maintainingpolypeptide backbone structure (eg., sheet or helical conformation) nearthe substitution, charge or hydrophobicity of the molecule at the targetsite, or bulk of a specific side chain. The following substitutions aregenerally expected to produce the greatest changes in proteinproperties: (a) a hydrophilic residue (e.g., seryl or threonyl) issubstituted for (or by) a hydrophobic residue (e.g., leucyl, isoleucyl,phenylalanyl, valyl or alanyl); (b) a cysteine or proline is substitutedfor (or by) any other residue; (c) a residue having an electropositiveside chain (e.g. lysyl, arginyl or histadyl) is substituted for (or by)an electronegative residue (eg., glutamyl or aspartyl); or (d) a residuehaving a bulky side chain (e.g., phenylalanine) is substituted for (orby) one lacking a side chain (e.g. glycine).

Variant MATER encoding sequences may be produced by standard DNAmutagenesis techniques, for example, M13 primer mutagenesis. Details ofthese techniques are provided in Sambrook et al. (In Molecular Cloning:A Laboratory Manual, CSHL, New York, 1989), Ch. 15. By the use of suchtechniques, variants may be created that differ in minor ways from thehuman MATER sequences disclosed. DNA molecules and nucleotide sequencesthat are derivatives of those specifically disclosed herein, and whichdiffer from those disclosed by the deletion, addition, or substitutionof nucleotides while still encoding a protein that has at least 82%sequence identity with the human MATER encoding sequence disclosed (SEQID NOs: 1 and 3), are comprehended by this disclosure. Also comprehendedare more closely related nucleic acid molecules that share at least 80%,at least 85%, at least 90%, at least 95%, or at least 98% nucleotidesequence homology with the disclosed Mater sequences. In their mostsimple form, such variants may differ from the disclosed sequences byalteration of the coding region to fit the codon usage bias of theparticular organism into which the molecule is to be introduced. Thefull length human Mater cDNA (SEQ ID NO: 23) is also encompassed in thedisclosure, and as a molecule that comprises both fragment 1 (SEQ IDNO: 1) and fragment 2 (SEQ ID NO: 3), and encoding the human MATERprotein with described physical characteristics and biologicalproperties.

Alternatively, the coding region may be altered by taking advantage ofthe degeneracy of the genetic code to alter the coding sequence suchthat, while the nucleotide sequence is substantially altered, itnevertheless encodes a protein having an amino acid sequencesubstantially similar to the disclosed human MATER protein sequences.For example, because of the degeneracy of the genetic code, fournucleotide codon triplets—(GCT, GCG, GCC and GCA)— code for alanine. Thecoding sequence of any specific alanine residue within the human MATERprotein, therefore, could be changed to any of these alternative codonswithout affecting the amino acid composition or characteristics of theencoded protein. Based upon the degeneracy of the genetic code, variantDNA molecules may be derived from the cDNA and gene sequences disclosedherein using standard DNA mutagenesis techniques as described above, orby synthesis of DNA sequences. Thus, this disclosure also encompassesnucleic acid sequences that encode a MATER protein, but which vary fromthe disclosed nucleic acid sequences by virtue of the degeneracy of thegenetic code.

Variants of the MATER protein may also be defined in terms of theirsequence identity with the prototype human MATER protein. As describedabove, human MATER proteins share at least 65%, at least 70%, at least75%, at least 80%, at least 90%, at least 95%, or at least 98% aminoacid sequence identity with the human MATER protein (SEQ ID NO: 24) orfragments disclosed herein (such as SEQ ID NOs: 2 and 4). Nucleic acidsequences that encode such proteins/fragments readily may be determinedsimply by applying the genetic code to the amino acid sequence of anMATER protein or fragment, and such nucleic acid molecules may readilybe produced by assembling oligonucleotides corresponding to portions ofthe sequence.

Nucleic acid molecules that are derived from the human Mater cDNAnucleic acid sequences disclosed include molecules that hybridize understringent conditions to the disclosed prototypical Mater nucleic acidmolecules, or fragments thereof. Stringent conditions are hybridizationat 65° C. in 6×SSC, 5× Denhardt's solution, 0.5% SDS and 100 μg/mlsheared salmon testes DNA, followed by 15–30 minute sequential washes in2×SSC, 0.5% SDS, followed by 1×SSC, 0.5% SDS and finally 0.2×SSC, 0.5%SDS, at 65° C.

Low stringency hybridization conditions (to detect less closely relatedhomologs) are performed as described above but at 50° C. (bothhybridization and wash conditions); however, depending on the strengthof the detected signal, the wash steps may be terminated after the first2×SSC wash.

Human Mater nucleic acid encoding molecules (including the cDNA shown inSEQ ID NO: 23 and fragments shown in SEQ ID NOs: 1 and 3, and nucleicacids comprising these sequences), and orthologs and homologs of thesesequences, may be incorporated into transformation or expressionvectors.

EXAMPLE 8 Expression of MATER Protein

With the provision of human Mater cDNA sequence fragments, and methodsfor determining and cloning the full length human Mater cDNA, theexpression and purification of the MATER protein by standard laboratorytechniques is now enabled. Purified human MATER protein may be used forfunctional analyses, antibody production, diagnostics, and patienttherapy. Furthermore, the DNA sequence of the Mater cDNA can bemanipulated in studies to understand the expression of the gene and thefunction of its product. Mutant forms of the human MATER may be isolatedbased upon information contained herein, and may be studied in order todetect alteration in expression patterns in terms of relativequantities, cellular localization, tissue specificity and functionalproperties of the encoded mutant MATER protein. Partial or full-lengthcDNA sequences, which encode for the subject protein, may be ligatedinto bacterial expression vectors. Methods for expressing large amountsof protein from a cloned gene introduced into Escherichia coli (E. coli)may be utilized for the purification, localization and functionalanalysis of proteins. For example, fusion proteins consisting of aminoterminal peptides encoded by a portion of the E. coli lacZ or trpE genelinked to MATER proteins may be used to prepare polyclonal andmonoclonal antibodies against these proteins. Thereafter, theseantibodies may be used to purify proteins by immunoaffinitychromatography, in diagnostic assays to quantitate the levels of proteinand to localize proteins in tissues and individual cells byimmunofluorescence. Such antibodies may be specific for epitope tags,which can be added to the expression construct for identification an/orpurification purposes.

Intact native protein may also be produced in E. coli in large amountsfor functional studies. Methods and plasmid vectors for producing fusionproteins and intact native proteins in bacteria are described inSambrook et al. (Sambrook et al., In Molecular Cloning: A LaboratoryManual, Ch. 17, CSHL, New York, 1989). Such fusion proteins may be madein large amounts, are easy to purify, and can be used to elicit antibodyresponse. Native proteins can be produced in bacteria by placing astrong, regulated promoter and an efficient ribosome binding siteupstream of the cloned gene. If low levels of protein are produced,additional steps may be taken to increase protein production; if highlevels of protein are produced, purification is relatively easy.Suitable methods are presented in Sambrook et al. (In Molecular Cloning:A Laboratory Manual, CSHL, New York, 1989) and are well known in theart. Often, proteins expressed at high levels are found in insolubleinclusion bodies. Methods for extracting proteins from these aggregatesare described by Sambrook et al. (In Molecular Cloning: A LaboratoryManual, Ch. 17, CSHL, New York, 1989). Vector systems suitable for theexpression of lacZ fusion genes include the pUR series of vectors(Ruther and Muller-Hill, EMBO J. 2:1791, 1983), pEX1-3 (Stanley andLuzio, EMBO J. 3:1429, 1984) and pMR100 (Gray et al., Proc. Natl. Acad.Sci. USA 79:6598, 1982). Vectors suitable for the production of intactnative proteins include pKC30 (Shimatake and Rosenberg, Nature 292:128,1981), pKK177-3 (Amann and Brosius, Gene 40:183, 1985) and pET-3(Studiar and Moffatt, J. Mol. Biol. 189:113, 1986). MATER fusionproteins may be isolated from protein gels, lyophilized, ground into apowder and used as an antigen. The DNA sequence can also be transferredfrom its existing context to other cloning vehicles, such as otherplasmids, bacteriophages, cosmids, animal viruses and yeast artificialchromosomes (YACs) (Burke et al., Science 236:806812, 1987). Thesevectors may then be introduced into a variety of hosts including somaticcells, and simple or complex organisms, such as bacteria, fungi(Timberlake and Marshall, Science 244:1313–1317, 1989), invertebrates,plants, and animals (Pursel et al., Science 244:1281–1288, 1989), whichcells or organisms are rendered transgenic by the introduction of theheterologous Mater cDNA.

For expression in mammalian cells, the cDNA sequence may be ligated toheterologous promoters, such as the simian virus (SV) 40 promoter in thepSV2 vector (Mulligan and Berg, Proc. Natl. Acad Sci. USA 78:2072–2076,1981), and introduced into cells, such as monkey COS-1 cells (Gluzman,Cell 23:175–182, 1981), to achieve transient or long-term expression.The stable integration of the chimeric gene construct may be maintainedin mammalian cells by biochemical selection, such as neomycin (Southernand Berg, J. Mol. Appl. Genet. 1:327–341, 1982) and mycophenolic acid(Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78:2072–2076, 1981).

DNA sequences can be manipulated with standard procedures such asrestriction enzyme digestion, fill-in with DNA polymerase, deletion byexonuclease, extension by terminal deoxynucleotide transferase, ligationof synthetic or cloned DNA sequences, site-directed sequence-alterationvia single-stranded bacteriophage intermediate or with the use ofspecific oligonucleotides in combination with nucleic acidamplification.

The cDNA sequence (or portions derived from it) or a mini gene (a cDNAwith an intron and its own promoter) may be introduced into eukaryoticexpression vectors by conventional techniques. These vectors aredesigned to permit the transcription of the cDNA in eukaryotic cells byproviding regulatory sequences that initiate and enhance thetranscription of the cDNA and ensure its proper splicing andpolyadenylation. Vectors containing the promoter and enhancer regions ofthe SV40 or long terminal repeat (LTR) of the Rous Sarcoma virus andpolyadenylation and splicing signal from SV40 are readily available(Mulligan et al., Proc. Natl. Acad Sci USA 78:1078–2076, 1981; Gorman etal, Proc. Natl. Acad. Sci USA 78:6777–6781, 1982). The level ofexpression of the cDNA can be manipulated with this type of vector,either by using promoters that have different activities (for example,the baculovirus pAC373 can express cDNAs at high levels in S. frugiperdacells (Summers and Smith, In Genetically Altered Viruses and theEnvironment, Fields et al. (Eds.) 22:319–328, CSHL Press, Cold SpringHarbor, N.Y., 1985) or by using vectors that contain promoters amenableto modulation, for example, the glucocorticoid-responsive promoter fromthe mouse mammary tumor virus (Lee et al., Nature 294:228, 1982). Theexpression of the cDNA can be monitored in the recipient cells 24 to 72hours after introduction (transient expression).

In addition, some vectors contain selectable markers such as the gpt(Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78:2072–2076, 1981) orneo (Southern and Berg, J. Mol. Appl. Genet. 1:327–341, 1982) bacterialgenes. These selectable markers permit selection of transfected cellsthat exhibit stable, long-term expression of the vectors (and thereforethe cDNA). The vectors can be maintained in the cells as episomal,freely replicating entities by using regulatory elements of viruses,such as papilloma (Sarver et al., Mol. Cell Biol. 1:486–496, 1981) orEpstein-Barr (Sugden et al., Mol. Cell Biol. 5:410–413, 1985).Alternatively, one can also produce cell lines that have integrated thevector into genomic DNA. Both of these types of cell lines produce thegene product on a continuous basis. One can also produce cell lines canalso produced that have amplified the number of copies of the vector(and therefore of the cDNA as well) to create cell lines that canproduce high levels of the gene product (Alt et al., J. Biol. Chem.253:1357–1370, 1978).

The transfer of DNA into eukaryotic, in particular human or othermammalian cells, is now a conventional technique. Recombinant expressionvectors can be introduced into the recipient cells as pure DNA(transfection) by, for example, precipitation with calcium phosphate(Graham and vander Eb, Virology 52:466, 1973) or strontium phosphate(Brash et al, Mol. Cell Biol. 7:2013, 1987), electroporation (Neumann etal., EMBO J 1:841, 1982), lipofection (Feigner et al, Proc. Natl. Acad.Sci USA 84:7413, 1987), DEAE dextran (McCuthan et al., J. Natl. CancerInst. 41:351, 1968), microinjection (Mueller et al., Cell 15:579, 1978),protoplast fusion (Schafner, Proc. Natl. Acad. Sci. USA 77:2163–2167,1980), or pellet guns (Klein et al, Nature 327:70, 1987). Alternatively,the cDNA, or fragments thereof, can be introduced by infection withvirus vectors. Systems are developed that use, for example, retroviruses(Bernstein et al., Gen. Engr'g 7:235, 1985), adenoviruses (Ahmad et al.,J. Virol. 57:267, 1986), or Herpes virus (Spaete et al., Cell 30:295,1982). Techniques of use in packaging long transcripts can be found inKochanek et al (Proc. Natl. Acad. Sci. USA 93:5731–5739, 1996) Parks etal (Proc. Natl. Acad. Sci. USA 93:13565–13570, 1996) and Parks andGraham (J. Virol. 71:3293–3298, 1997). MATER encoding sequences can alsobe delivered to target cells in vitro via non-infectious systems, forinstance liposomes.

These eukaryotic expression systems can be used for studies of MATERencoding nucleic acids and mutant forms of these molecules, the MATERprotein and mutant forms of this protein. Such uses include, forexample, the identification of regulatory elements located in the 5′region of the Mater gene on genomic clones that can be isolated fromhuman genomic DNA libraries using the information contained herein. Theeukaryotic expression systems also may be used to study the function ofthe normal complete protein, specific portions of the protein, or ofnaturally occurring or artificially produced mutant proteins.

Using the above techniques, expression vectors containing the Mater genesequence or cDNA, or fragments or variants or mutants thereof, can beintroduced into human cells, mammalian cells from other species ornon-mammalian cells, as desired. The choice of cell is determined by thepurpose of the treatment. For example, monkey COS cells (Gluzman, Cell23:175–182, 1981) that produce high levels of the SV40 T antigen andpermit the replication of vectors containing the SV40 origin ofreplication may be used. Similarly, Chinese hamster ovary (CHO), mouseNIH 3T3 fibroblasts or human fibroblasts or lymphoblasts (as describedherein) may be used.

Embodiments described herein thus encompass recombinant vectors thatcomprise all or part of a MATER encoding sequence, such as the Matergene or cDNA or variants thereof, for expression in a suitable host. TheMater DNA is operatively linked in the vector to an expression controlsequence in the recombinant DNA molecule so that the MATER polypeptidecan be expressed. The expression control sequence may be selected fromthe group consisting of sequences that control the expression of genesof prokaryotic or eukaryotic cells and their viruses and combinationsthereof. The expression control sequence may be specifically selectedfrom the group consisting of the lac system, the trp system, the tacsystem, the trc system, major operator and promoter regions of phagelambda, the control region of fd coat protein, the early and latepromoters of SV40, promoters derived from polyoma, adenovirus,retrovirus, baculovirus and simian virus, the promoter for3-phosphoglycerate kinase, the promoters of yeast acid phosphatase, thepromoter of the yeast alpha-mating factors and combinations thereof.

The host cell, which may be transfected with a vector, may be selectedfrom the group consisting of E. coli, Pseudomonas, Bacillus subtilis,Bacillus stearothermophilus or other bacilli; other bacteria; yeast;fungi; insect; mouse or other animal; or plant hosts; or human tissuecells.

It is appreciated that for mutant or variant Mater DNA sequences,similar systems are employed to express and produce the mutant product.

EXAMPLE 9 Production of an Antibody to MATER Protein

Monoclonal or polyclonal antibodies may be produced to either the normalMATER protein or mutant forms of this protein. Optimally, antibodiesraised against the MATER protein would specifically detect the MATERprotein. That is, such antibodies would recognize and bind the MATERprotein and would not substantially recognize or bind to other proteinsfound in human cells. Antibodies the human MATER protein may recognizeMATER from other species, such as murine MATER, and vice versa.

The determination that an antibody specifically detects the MATERprotein is made by any one of a number of standard immunoassay methods;for instance, the Western blotting technique (Sambrook et al., InMolecular Cloning: A Laboratory Manual, CSHL, New York, 1989). Todetermine that a given antibody preparation (such as one produced in amouse) specifically detects the MATER protein by Western blotting, totalcellular protein is extracted from human cells (for example,lymphocytes) and electrophoresed on a sodium dodecylsulfate-polyacrylamide gel. The proteins are then transferred to amembrane (for example, nitrocellulose or PVDF) by Western blotting, andthe antibody preparation is incubated with the membrane. After washingthe membrane to remove non-specifically bound antibodies, the presenceof specifically bound antibodies is detected by the use of (by way ofexample) an anti-mouse antibody conjugated to an enzyme such as alkalinephosphatase. Application of an alkaline phosphatase substrate5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium results inthe production of a dense blue compound by immunolocalized alkalinephosphatase. Antibodies that specifically detect the MATER protein will,by this technique, be shown to bind to the MATER protein band (whichwill be localized at a given position on the gel determined by itsmolecular weight, which is approximately 125 kDa based on gel-mobilityestimation for murine MATER. Non-specific binding of the antibody toother proteins may occur and may be detectable as a weak signal on theWestern blot. The non-specific nature of this binding will be recognizedby one skilled in the art by the weak signal obtained on the Westernblot relative to the strong primary signal arising from the specificantibody-MATER protein binding.

Substantially pure MATER protein suitable for use as an immunogen isisolated from the transfected or transformed cells as described above.The concentration of protein in the final preparation is adjusted, forexample, by concentration on an Amicon (Millipore, Bedford, Mass.) orsimilar filter device, to the level of a few micrograms per milliliter.Monoclonal or polyclonal antibody to the protein can then be prepared asfollows:

A. Monoclonal Antibody Production by Hybridoma Fusion

Monoclonal antibody to epitopes of the MATER protein identified andisolated as described can be prepared from murine hybridomas accordingto the classical method of Kohler and Milstein (Nature 256:495–497,1975) or derivative methods thereof. Briefly, a mouse is repetitivelyinoculated with a few micrograms of the selected protein over a periodof a few weeks. The mouse is then sacrificed, and the antibody-producingcells of the spleen isolated. The spleen cells are fused with mousemyeloma cells using polyethylene glycol, and the excess un-fused cellsdestroyed by growth of the system on selective media comprisingaminopterin (HAT media). Successfully fused cells are diluted andaliquots of the dilution placed in wells of a microtiter plate, wheregrowth of the culture is continued. Antibody-producing clones areidentified by detection of antibody in the supernatant fluid of thewells by immunoassay procedures, such as ELISA, as originally describedby Engvall (Enzymol. 70(A):419–439, 1980), and derivative methodsthereof. Selected positive clones can be expanded and their monoclonalantibody product harvested for use. Detailed procedures for monoclonalantibody production are described in Harlow and Lane (Antibodies, ALaboratory Manual, CSHL, New York, 1988).

B. Polyclonal Antibody Production by Immunization

Polyclonal antiserum containing antibodies to heterogeneous epitopes ofa single protein can be prepared by immunizing suitable animals with theexpressed protein (Example 8), which optionally can be modified toenhance immunogenicity. Effective polyclonal antibody production isaffected by many factors related both to the antigen and the hostspecies. For example, small molecules tend to be less immunogenic thanothers and may require the use of carriers and adjuvant, examples ofwhich are known. Also, host animals vary in response to site ofinoculations and dose, with either inadequate or excessive doses ofantigen resulting in low titer antisera. A series of small doses (nglevel) of antigen administered at multiple intradermal sites appear tobe most reliable. An effective immunization protocol for rabbits can befound in Vaitukaitis et al. (J. Clin. Endocrinol. Metab. 33:988–991,1971).

Booster injections can be given at regular intervals, and antiserumharvested when antibody titer thereof begins to fall, as determinedsemi-quantitatively (for example, by double immunodiffusion in agaragainst known concentrations of the antigen). See, for example,Ouchterlony et al. (In Handbook of Experimental Immunology, Wier, D.(ed.) chapter 19. Blackwell, 1973). Plateau concentration of antibody isusually in the range of about 0.1 to 0.2 mg/ml of serum (about 12 μM).Affinity of the antisera for the antigen is determined by preparingcompetitive binding curves, as described, for example, by Fisher (Manualof Clinical Immunology, Ch. 42, 1980).

C. Antibodies Raised against Synthetic Peptides

A third approach to raising antibodies against the MATER protein is touse synthetic peptides synthesized on a commercially available peptidesynthesizer based upon the predicted amino acid sequence of the MATERprotein.

By way of example only, mouse MATER C-terminal peptide (residues 1093through 1111 of SEQ ID NO: 6) was conjugated with KLH to immunize thefemale rabbits (two) every two-weeks. Starting from the thirdimmunization, a small amount (˜3 ml) of blood was collected from theimmunized rabbits to examine the titer of the anti-peptide antibodiesusing the peptide as antigen and ELISA method. Immunizations continueduntil the antibodies reached maximal titers, which occurred in about tenimmunizations, and then the rabbits were sacrificed to bleed inpreparation for the sera. The resultant preparation was used both tocharacterize murine MATER (Example 1) and human MATER (Example 4).

D. Antibodies Raised by Injection of MATER Encoding Sequence

Antibodies may be raised against the MATER protein by subcutaneousinjection of a recombinant DNA vector that expresses the MATER proteininto laboratory animals, such as mice. Delivery of the recombinantvector into the animals may be achieved using a hand-held form of theBiolistic system (Sanford et al., Particulate Sci. Technol. 5:27–37,1987), as described by Tang et al. (Nature 356:152–154, 1992).Expression vectors suitable for this purpose may include those thatexpress the MATER encoding sequence under the transcriptional control ofeither the human β-actin promoter or the cytomegalovirus (CMV) promoter.

Antibody preparations prepared according to these protocols are usefulin quantitative immunoassays which determine concentrations ofantigen-bearing substances in biological samples; they are also usedsemi-quantitatively or qualitatively to identify the presence of antigenin a biological sample.

EXAMPLE 10 DNA-Based Diagnosis

The Mater sequence information presented herein can be used in the areaof genetic testing for predisposition to reduced fertility orinfertility, such as autoimmune infertility, owing to defects in Mater,such as deletion, duplication or mutation. The gene sequence of theMater gene, including intron-exon boundaries is also useful in suchdiagnostic methods. Individuals carrying mutations in the Mater gene (ora portion thereof), or having duplications or heteroygous or homozygousdeletions of the Mater gene, may be detected at the DNA level with theuse of a variety of techniques. For such a diagnostic procedure, abiological sample of the subject, which biological sample containseither DNA or RNA derived from the subject, is assayed for a mutated,duplicated or deleted Mater gene. Suitable biological samples includesamples containing genomic DNA or RNA obtained from body cells, such asthose present in peripheral blood, urine, saliva, tissue biopsy,surgical specimen, amniocentesis samples and autopsy material. Thedetection in the biological sample of either a mutant Mater gene, amutant Mater RNA, or a duplicated or homozygously or heterozygouslydeleted Mater gene, may be performed by a number of methodologies,examples of which are discussed below.

One embodiment of such detection techniques for the identification ofunknown mutations is the amplification (e.g., polymerase chain reactionamplification) of reverse transcribed RNA (RT-PCR) isolated from asubject, followed by direct DNA sequence determination of the products.The presence of one or more nucleotide differences between the obtainedsequence and the prototypical Mater cDNA sequence, and especially,differences in the ORF portion of the nucleotide sequence, are taken asindicative of a potential Mater gene mutation.

Alternatively, DNA extracted from a biological sample may be useddirectly for amplification. Direct amplification from genomic DNA wouldbe appropriate for analysis of the entire Mater gene includingregulatory sequences located upstream and downstream from the openreading frame, or intron/exon borders. Reviews of direct DNA diagnosishave been presented by Caskey (Science 236:1223–1228, 1989) and byLandegren et al. (Science 242:229–237, 1989).

Other mutation scanning techniques appropriate for detecting unknownwithin amplicons derived from DNA or cDNA could also be performed. Thesetechniques include direct sequencing (without sequencing), single-strandconformational polymorphism analysis (SSCP) (for instance, see Hongyo etal., Nucleic Acids Res. 21:3637–3642, 1993), chemical cleavage(including HOT cleavage) (Bateman et al., Am. J. Med. Genet. 45:233–240,1993; reviewed in Ellis et al., Hum. Mutat 11:345–353, 1998), denaturinggradient gel electrophoresis (DGGE), ligation amplification mismatchprotection (LAMP), and enzymatic mutation scanning (Taylor and Deeble,Genet. Anal. 14:181–186, 1999), followed by direct sequencing ofamplicons with putative sequence variations.

Further studies of Mater genes isolated from female subjects displayinginfertility, particularly autoimmune infertility subjects, or theirrelatives, may reveal particular mutations, genomic amplifications, ordeletions, which occur at a high frequency within this population ofindividuals. In such case, rather than sequencing the entire Mater gene,DNA diagnostic methods can be designed to specifically detect the mostcommon, or most closely disease-linked, MATER defects.

The detection of specific DNA mutations may be achieved by methods suchas hybridization using allele specific oligonucleotides (ASOs) (Wallaceet al., CSHL Symp. Quant. Biol. 51:257–261, 1986), direct DNA sequencing(Church and Gilbert Proc. Natl. Acad Sci. USA 81:1991–1995, 1988), theuse of restriction enzymes (Flavell et al., Cell 15:25–41, 1978; Geeveret al., 1981), discrimination on the basis of electrophoretic mobilityin gels with denaturing reagent (Myers and Maniatis, Cold Spring HarborSymp. Quant. Biol. 51:275–284, 1986), RNase protection (Myers et al.,Science 230:1242–1246, 1985), chemical cleavage (Cotton et al., Proc.Natl. Acad. Sci. USA 85:4397–4401, 1985), and the ligase-mediateddetection procedure (Landegren et al., Science 241:1077–1080, 1988).

Oligonucleotides specific to normal or mutant sequences are chemicallysynthesized using commercially available machines. Theseoligonucleotides are then labeled radioactively with isotopes (such as³²P) or non-radioactively, with tags such as biotin (Ward and Langer,Proc. Natl. Acad. Sci. USA 78:6633–6657, 1981), and hybridized toindividual DNA samples immobilized on membranes or other solid supportsby dot-blot or transfer from gels after electrophoresis. These specificsequences are visualized by methods such as autoradiography orfluorometric (Landegren et al., Science 242:229–237, 1989) orcalorimetric reactions (Gebeyehu et at, Nucleic Acids Res. 15:4513–4534,1987). Using an ASO specific for a normal allele, the absence ofhybridization would indicate a mutation in the particular region of thegene, or deleted Mater gene. In contrast, if an ASO specific for amutant allele hybridizes to a clinical sample, this would indicate thepresence of a mutation in the region defined by the ASO.

Sequence differences between normal and mutant forms of the Mater genemay also be revealed by the direct DNA sequencing method of Church andGilbert (Proc. Natl. Acad. Sci. USA 81:1991–1995, 1988). Cloned DNAsegments may be used as probes to detect specific DNA segments. Thesensitivity of this method is greatly enhanced when combined withnucleic acid amplification, e.g., PCR (Wrichnik et al., Nucleic AcidsRes. 15:529–542, 1987; Wong et al., Nature 330:384–386, 1987; Stoflet etal., Science 239:491–494, 1988). In this approach, a sequencing primerthat lies within the amplified sequence is used with double-stranded PCRproduct or single-stranded template generated by a modified PCR. Thesequence determination is performed by conventional procedures withradiolabeled nucleotides or by automatic sequencing procedures withfluorescent tags.

Sequence alterations may occasionally generate fortuitous restrictionenzyme recognition sites or may eliminate existing restriction sites.Changes in restriction sites are revealed by the use of appropriateenzyme digestion followed by conventional gel-blot hybridization(Southern, J. Mol. Biol. 98:503–517, 1975). DNA fragments carrying therestriction site (either normal or mutant) are detected by theirreduction in size or increase in corresponding restriction fragmentnumbers.

Genomic DNA samples may also be amplified by PCR prior to treatment withthe appropriate restriction enzyme; fragments of different sizes arethen visualized under UV light in the presence of ethidium bromide aftergel electrophoresis.

Genetic testing based on DNA sequence differences may be achieved bydetection of alteration in electrophoretic mobility of DNA fragments ingels, with or without denaturing reagent. Small sequence deletions andinsertions can be visualized by high-resolution gel electrophoresis. Forexample, a PCR product with small deletions is clearly distinguishablefrom a normal sequence on an 8% non-denaturing polyacrylamide gel (WO91/10734; Nagamine et al., Am. J. Hum. Genet. 45:337–339, 1989). DNAfragments of different sequence compositions may be distinguished ondenaturing formamide gradient gels in which the mobilities of differentDNA fragments are retarded in the gel at different positions accordingto their specific “partial-melting” temperatures (Myers et al., Science230:1242–1246, 1985). Alternatively, a method of detecting a mutationcomprising a single base substitution or other small change could bebased on differential primer length in a PCR. For example, an invariantprimer could be used in addition to a primer specific for a mutation.The PCR products of the normal and mutant genes can then bedifferentially detected in acrylamide gels.

In addition to conventional gel-electrophoresis and blot-hybridizationmethods, DNA fragments may also be visualized by methods where theindividual DNA samples are not immobilized on membranes. The probe andtarget sequences may be both in solution, or the probe sequence may beimmobilized (Saiki et al., Proc. Nat. Acad. Sci. USA 86:6230–6234,1989). A variety of detection methods, such as autoradiography involvingradioisotopes, direct detection of radioactive decay (in the presence orabsence of scintillant), spectrophotometry involving calorigenicreactions and fluorometry involved fluorogenic reactions, may be used toidentify specific individual genotypes.

If multiple mutations are encountered frequently in the Mater gene, asystem capable of detecting such multiple mutations likely will bedesirable. For example, a nucleic acid amplification reaction withmultiple, specific oligonucleotide primers and hybridization probes maybe used to identify all possible mutations at the same time (Chamberlainet al., Nucl. Acids Res. 16:1141–1155, 1988). The procedure may involveimmobilized sequence-specific oligonucleotide probes (Saiki et al, Proc.Nat. Acad. Sci. USA 86:6230–6234, 1989).

EXAMPLE 11 Quantitation of MATER Protein

An alternative method of diagnosing Mater gene deletion, amplification,or mutation is to quantitate the level of MATER protein in the cells ofa subject. This diagnostic tool would be useful for detecting reducedlevels of the MATER protein that result from, for example, mutations inthe promoter regions of the Mater gene or mutations within the codingregion of the gene that produce truncated, non-functional or unstablepolypeptides, as well as from deletions of the entire Mater gene.Alternatively, duplications of the Mater gene may be detected as anincrease in the expression level of this protein. The determination ofreduced or increased MATER protein levels would be an alternative orsupplemental approach to the direct determination of Mater genedeletion, duplication or mutation status by the methods outlined above.

The availability of antibodies specific to the MATER protein willfacilitate the quantitation of cellular MATER protein by one of a numberof immunoassay methods, which are well known in the art and arepresented in Harlow and Lane (Antibodies, A Laboratory Manual, CSHL, NewYork 1988).

For the purposes of quantitating the MATER protein, a biological sampleof the subject, which sample includes cellular proteins, is required.Such a biological sample may be obtained from body cells, such as thosepresent in peripheral blood, urine, saliva, tissue biopsy, amniocentesissamples, surgical specimens and autopsy material. In particular, femalereproductive cells (e.g. ova) or embryos are appropriate samples.Quantitation of MATER protein is achieved by immunoassay and compared tolevels of the protein found in healthy cells (e.g. cells from a femaleknown not to suffer from decreased fertility). A significant (e.g., 10%or greater, for instance, 20%, 25%, 30%, 50% or more) reduction in theamount of MATER protein in the cells of a subject compared to the amountof MATER protein found in normal human cells would be taken as anindication that the subject may have deletions or mutations in the Matergene locus, whereas a significant (e.g., 10% or greater, for instance,20%, 25%, 30%, 50% or more) increase would indicate that a duplicationor enhancing mutation had occurred.

EXAMPLE 12 Detection of Serum Antibody Against MATER Protein

The MATER family of proteins was first identified as autoantibodiesinvolved in the pathogenesis of certain cases of autoimmune infertility.With the provision herein of human MATER protein sequences and encodingnucleic acids, methods for the detection and diagnosis of such fertilityfailure are now enabled.

Autoantibodies that recognize an epitope of the human MATER protein canbe detected in samples from a subject, for instance serum or otherfluid, using known immunological techniques. The presence of suchautoantibodies (e.g., circulating autoantibodies specific for a MATERepitope) indicates that the subject suffers from MATER-mediatedinfertility or reduced fertility, or has an increased susceptibility tosuffer from one of these conditions.

Many techniques are commonly known in the art for the detection andquantification of antigen. Most commonly, the purified antigen will bebound to a substrate, the antibody of the sample will bind via its Fabportion to this antigen, the substrate will then be washed and a second,labeled antibody will then be added which will bind to the Fc portion ofthe antibody that is the subject of the assay. The second, labeledantibody will be species specific, i.e., if the serum is from a human,the second, labeled antibody will be anti-human-IgG antibody. Thespecimen will then be washed and the amount of the second, labeledantibody that has been bound will be detected and quantified by standardmethods.

Examples of methods for the detection of antibodies in biologicalsamples, including methods employing dip strips or other immobilizedassay devices, are disclosed for instance in the following patents: U.S.Pat. No. 5,965,356 (Herpes simplex virus type specific seroassay); U.S.Pat. No. 6,114,179 (Method and test kit for detection of antigens and/orantibodies); U.S. Pat. No. 6,077,681 (Diagnosis of motor neuropathy bydetection of antibodies); U.S. Pat. No. 6,057,097 (Marker forpathologies comprising an auto-immune reaction and/or for inflammatorydiseases); and U.S. Pat. No. 5,552,285 (Immunoassay methods,compositions and kits for antibodies to oxidized DNA bases).

EXAMPLE 13 Suppression of MATER Expression

A reduction of MATER protein expression in a transgenic cell may beobtained by introducing into cells an antisense construct based on theMater encoding sequence, including the human Mater cDNA or fragmentsthereof (for instance, the cDNA fragments shown in SEQ ID NO: 1 and 3)or gene sequence or flanking regions thereof. For antisense suppression,a nucleotide sequence from an MATER encoding sequence, e.g. all or aportion of the Mater cDNA or gene, is arranged in reverse orientationrelative to the promoter sequence in the transformation vector. Otheraspects of the vector may be chosen as discussed above (Example 8).

The introduced sequence need not be the full length human Mater cDNA(SEQ ID NO: 23) or gene, and need not be exactly homologous to theequivalent sequence found in the cell type to be transformed. Thus,portions or fragments of the murine cDNA (SEQ ID NO: 5) could also beused to knock out expression of the human Mater gene. Generally,however, where the introduced sequence is of shorter length, a higherdegree of homology to the native Mater sequence will be needed foreffective antisense suppression. The introduced antisense sequence inthe vector may be at least 30 nucleotides in length, and improvedantisense suppression typically will be observed as the length of theantisense sequence increases. The length of the antisense sequence inthe vector advantageously may be greater than 100 nucleotides, and canbe up to about the full length of the human Mater cDNA or gene. Forsuppression of the Mater gene itself, transcription of an antisenseconstruct results in the production of RNA molecules that are thereverse complement of mRNA molecules transcribed from the endogenousMater gene in the cell.

Although the exact mechanism by which antisense RNA molecules interferewith gene expression has not been elucidated, it is believed thatantisense RNA molecules bind to the endogenous mRNA molecules andthereby inhibit translation of the endogenous mRNA.

Suppression of endogenous MATER expression can also be achieved usingribozymes. Ribozymes are synthetic RNA molecules that possess highlyspecific endoribonuclease activity. The production and use of ribozymesare disclosed in U.S. Pat. No. 4,987,071 to Cech and U.S. Pat. No.5,543,508 to Haselhoff. The inclusion of ribozyme sequences withinantisense RNAs may be used to confer RNA cleaving activity on theantisense RNA, such that endogenous mRNA molecules that bind to theantisense RNA are cleaved, which in turn leads to an enhanced antisenseinhibition of endogenous gene expression.

Finally, dominant negative mutant forms of MATER may be used to blockendogenous MATER activity.

EXAMPLE 14 MATER Knockout and Overexpression Transgenic Animals

Mutant organisms that under-express or over-express MATER protein areuseful for research. Such mutants allow insight into the physiologicaland/or pathological role of MATER in a healthy and/or pathologicalorganism. These mutants are “genetically engineered,” meaning thatinformation in the form of nucleotides has been transferred into themutant's genome at a location, or in a combination, in which it wouldnot normally exist. Nucleotides transferred in this way are said to be“non-native.” For example, a non-Mater promoter inserted upstream of anative Mater gene would be non-native. An extra copy of a Mater gene orother encoding sequence on a plasmid, transformed into a cell, would benon-native, whether that extra copy was Mater derived from the same or adifferent species.

Mutants may be, for example, produced from mammals, such as mice, thateither over-express or under-express MATER protein, or that do notexpress MATER at all. Over-expression mutants are made by increasing thenumber of MATER-encoding sequences (such as genes) in the organism, orby introducing an MATER-encoding sequence into the organism under thecontrol of a constitutive or inducible or viral promoter such as themouse mammary tumor virus (MMTV) promoter or the whey acidic protein(WAP) promoter or the metallothionein promoter. Mutants thatunder-express MATER may be made by using an inducible or repressiblepromoter, or by deleting the Mater gene, or by destroying or limitingthe function of the Mater gene, for instance by disrupting the gene bytransposon insertion.

Antisense genes may be engineered into the organism, under aconstitutive or inducible promoter, to decrease or prevent MATERexpression, as discussed above in Example 13.

A gene is “functionally deleted” when genetic engineering has been usedto negate or reduce gene expression to negligible levels. When a mutantis referred to in this application as having the Mater gene altered orfunctionally deleted, this refers to the Mater gene and to any orthologof this gene. When a mutant is referred to as having “more than thenormal copy number” of a gene, this means that it has more than theusual number of genes found in the wild-type organism, e.g. in thediploid mouse or human.

A mutant mouse over-expressing MATER may be made by constructing aplasmid having the Mater gene driven by a promoter, such as the mousemammary tumor virus (MMTV) promoter or the whey acidic protein (WAP)promoter. This plasmid may be introduced into mouse oocytes bymicroinjection. The oocytes are implanted into pseudopregnant females,and the litters are assayed for insertion of the transgene. Multiplestrains containing the transgene are then available for study.

WAP is quite specific for mammary gland expression during lactation, andMMTV is expressed in a variety of tissues including mammary gland,salivary gland and lymphoid tissues. Many other promoters might be usedto achieve various patterns of expression, e.g. the metallothioneinpromoter.

An inducible system may be created in which the subject expressionconstruct is driven by a promoter regulated by an agent that can be fedto the mouse, such as tetracycline. Such techniques are well known inthe art.

A mutant knockout animal (e.g., mouse) from which the Mater gene isdeleted or otherwise disabled can be made by removing coding regions ofthe Mater gene from embryonic stem cells. The methods of creatingdeletion mutations by using a targeting vector have been described (see,for instance, Thomas and Capecch, Cell 51:503–512, 1987). One specificexample of the production of a Mater null mouse is described above, inExample 1.

EXAMPLE 15 Gene Therapy

Gene therapy approaches for combating MATER-mediated fertility defectsin subjects, or for causing MATER-mediated infertility in subjects, arenow made possible.

Retroviruses have been considered the preferred vector for experimentsin gene therapy, with a high efficiency of infection and stableintegration and expression (Orkin et al., Prog. Med Genet. 7:130–142,1988). The full-length Mater gene or cDNA can be cloned into aretroviral vector and driven from either its endogenous promoter or, forinstance, from the retroviral LTR (long terminal repeat). Other viraltransfection systems may also be utilized for this type of approach,including adenovirus, adeno-associated virus (AAV) (McLaughlin et al.,J. Virol. 62:1963–1973, 1988), Vaccinia virus (Moss et al, Annu. Rev.Immunol. 5:305–324, 1987), Bovine Papilloma virus (Rasmussen et al,Methods Enzymol. 139:642–654, 1987) or members of the herpesvirus groupsuch as Epstein-Barr virus (Margolskee et al., Mol. Cell. Biol.8:2837–2847, 1988).

Recent developments in gene therapy techniques include the use ofRNA-DNA hybrid oligonucleotides, as described by Cole-Strauss, et al.(Science 273:1386–1389, 1996). This technique may allow forsite-specific integration of cloned sequences, thereby permittingaccurately targeted gene replacement.

In addition to delivery of Mater to cells using viral vectors, it ispossible to use non-infectious methods of delivery. For instance,lipidic and liposome-mediated gene delivery has recently been usedsuccessfully for transfection with various genes (for reviews, seeTempleton and Lasic, Mol. Biotechnol. 11:175–180, 1999; Lee and Huang,Crit. Rev. Ther. Drug Carrier Syst. 14:173–206; and Cooper, Semin.Oncol. 23:172–187, 1996). For instance, cationic liposomes have beenanalyzed for their ability to transfect monocytic leukemia cells, andshown to be a viable alternative to using viral vectors (de Lima et al.,Mol. Membr. Biol. 16:103–109, 1999). Such cationic liposomes can also betargeted to specific cells through the inclusion of, for instance,monoclonal antibodies or other appropriate targeting ligands (Kao etal., Cancer Gene Ther. 3:250–256, 1996).

EXAMPLE 16 Identification of Therapeutic Compounds

The human MATER molecules disclosed herein can be used to identify(screen for) compounds that are useful in influencing MATER-mediatedfertility in a mammal, either by blocking (inhibiting) the activity ofMATER (and thereby reducing fertility) or enhancing MATER activity (andthereby increasing fertility).

Such screening methods can include determining if a test compound bindsdirectly to or otherwise interacts with a MATER protein, or a variant orfragment of a MATER protein. Proteins that do bind to such a moleculeare select for further characterization.

In specific embodiments, the compound being tested for activity isapplied to a cell for instance a test cell (e.g., a developing oocyte orembryo of a mammal). The activity of the MATER protein in that test cellis then measured, for instance by determining whether the oocyte orembryo progresses beyond the two-cell stage. If application of the testcompound alters proportion of embryos that progress beyond two cells,then that compound is selected as a likely candidate for furthercharacterization. In particular examples, a test agent that opposes orinhibits a MATER activity is selected for further study, for example byexposing the agent to a mammalian female reproductive system in vivo, todetermine if in vivo fertility is inhibited. Such identified compoundsmay be useful as contraceptive agents.

Specific examples of compounds likely to be effective at inhibitingMATER activity, and therefore effective as contraceptives, includeantibodies specifically directed to epitopes of the MATER protein. Thegeneral concept of immunocontraceptives has been described (see, e.g.,U.S. Pat. Nos. 5,637,300 and 6,027,727, describing contraceptiveantibodies directed to proteins of the zona pellicida, and incorporatedherein by reference).

Alternatively, compounds that increase the proportion of embryos thatprogress beyond two cells (for instance, in an animal system known to bedefective for fertility) are selected for further study as possiblefertility enhancing agents. Similar screens can be used to identifycompounds that mimic the activity of MATER protein, for instance in aMater null animal.

In addition, it is suggested that MATER protein perform its functionwithin the cytoplasm through interacting with other unknown protein.Physical blockage of such interaction is expected to arrest the MATERfunction. Candidates for such interactions are being identified based oninteractions in a yeast two-hybrid system (Fields and Songs, Nature340:245, 1989). Once molecular domains for the protein interaction areknown, molecules can be designed directly based to specifically inhibitor otherwise interfere with MATER protein interactions with these otherproteins.

Compounds selected using these methods are comprehended by thisdisclosure.

EXAMPLE 17 Kits

Kits are provided which contain the necessary reagents for determiningMater gene copy number, such as probes or primers specific for the Matergene, as well as written instructions. Kits are also provided todetermine abnormal expression of Mater mRNA (i.e., containing probes) orMATER protein (i.e., containing a MATER-specific binding agent).Instructions provided in the diagnostic kits can include calibrationcurves or charts to compare with the determined (e.g., experimentallymeasured) values.

A. Kits for Detection of Mater Genomic Sequences

The nucleotide sequences disclosed herein, and fragments thereof, can besupplied in the form of a kit for use in detection of Mater genomicsequences and/or diagnosis of infertility or reduced fertility. In sucha kit, an appropriate amount of one or more of the Mater-specificoligonucleotide primers is provided in one or more containers. Theoligonucleotide primers may be provided suspended in an aqueous solutionor as a freeze-dried or lyophilized powder, for instance. Thecontainer(s) in which the oligonucleotide(s) are supplied can be anyconventional container that is capable of holding the supplied form, forinstance, microfuge tubes, ampoules, or bottles. In some applications,pairs of primers may be provided in pre-measured single use amounts inindividual, typically disposable, tubes or equivalent containers. Withsuch an arrangement, the sample to be tested for the presence of Matergenomic amplification can be added to the individual tubes and in vitroamplification carried out directly.

The amount of each oligonucleotide primer supplied in the kit can be anyappropriate amount, depending for instance on the market to which theproduct is directed. For instance, if the kit is adapted for research orclinical use, the amount of each oligonucleotide primer provided wouldlikely be an amount sufficient to prime several in vitro amplificationreactions. Those of ordinary skill in the art know the amount ofoligonucleotide primer that is appropriate for use in a singleamplification reaction. General guidelines may for instance be found inInnis et al. (PCR Protocols, A Guide to Methods and Applications,Academic Press, Inc., San Diego, Calif., 1990), Sambrook et al. (InMolecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989),and Ausubel et al. (In Current Protocols in Molecular Biology, JohnWiley & Sons, New York, 1998).

A kit may include more than two primers, in order to facilitate the PCRin vitro amplification of Mater sequences, for instance the Mater gene,specific exon(s) or other portions of the gene, or the 5′ or 3′ flankingregion thereof.

In some embodiments, kits may also include the reagents necessary tocarry out PCR in vitro amplification reactions, including, for instance,DNA sample preparation reagents, appropriate buffers (e.g., polymerasebuffer), salts (e.g., magnesium chloride), and deoxyribonucleotides(dNTPs). Written instructions may also be included.

Kits may in addition include either labeled or unlabeled oligonucleotideprobes for use in detection of the in vitro amplified Mater sequences.The appropriate sequences for such a probe will be any sequence thatfalls between the annealing sites of the two provided oligonucleotideprimers, such that the sequence the probe is complementary to isamplified during the in vitro amplification reaction.

It may also be advantageous to provided in the kit one or more controlsequences for use in the amplification reactions. The design ofappropriate positive control sequences is well known to one of ordinaryskill in the appropriate art.

B. Kits for Detection of Mater mRNA Expression

Kits similar to those disclosed above for the detection of Mater genomicsequences can be used to detect Mater mRNA expression levels. Such kitsmay include an appropriate amount of one or more of the oligonucleotideprimers for use in reverse transcription amplification reactions,similarly to those provided above, with art-obvious modifications foruse with RNA.

In some embodiments, kits for detection of Mater mRNA expression levelsmay also include the reagents necessary to carry out RT-PCR in vitroamplification reactions, including, for instance, RNA sample preparationreagents (including e.g. an RNAse inhibitor), appropriate buffers (e.g.,polymerase buffer), salts (e.g., magnesium chloride), anddeoxyribonucleotides (dNTPs). Written instructions may also be included.

Kits in addition may include either labeled or unlabeled oligonucleotideprobes for use in detection of the in vitro amplified target sequences.The appropriate sequences for such a probe will be any sequence thatfalls between the annealing sites of the two provided oligonucleotideprimers, such that the sequence the probe is complementary to isamplified during the PCR reaction.

It also may be advantageous to provided in the kit one or more controlsequences for use in the RT-PCR reactions. The design of appropriatepositive control sequences is well known to one of ordinary skill in theappropriate art.

Alternatively, kits may be provided with the necessary reagents to carryout quantitative or semi-quantitative Northern analysis of Mater mRNA.Such kits include, for instance, at least one Mater-specificoligonucleotide for use as a probe. This oligonucleotide may be labeledin any conventional way, including with a selected radioactive isotope,enzyme substrate, co-factor, ligand, chemiluminescent or fluorescentagent, hapten, or enzyme.

C. Kits for Detection of MATER Protein or Peptide Expression

Kits for the detection of MATER protein expression, for instance MATERat least one target (e.g., MATER) protein specific binding agent (e.g. apolyclonal or monoclonal antibody or antibody fragment) and may includeat least one control. The MATER protein specific binding agent andcontrol may be contained in separate containers. The kits may alsoinclude means for detecting MATER:agent complexes, for instance theagent may be detectably labeled. If the detectable agent is not labeled,it may be detected by second antibodies or protein A for example whichmay also be provided in some kits in one or more separate containers.Such techniques are well known.

Additional components in some kits include instructions for carrying outthe assay. Instructions will allow the tester to determine whether MATERexpression levels are altered, for instance in comparison to a controlsample. Reaction vessels and auxiliary reagents such as chromogens,buffers, enzymes, etc. may also be included in the kits.

By way of example only, an effective and convenient immunoassay kit suchas an enzyme-linked immunosorbent assay can be constructed to testanti-MATER antibody in human serum, as reported for detection ofnon-specific anti-ovarian antibodies (Wheatcroft et al, Clin. Exp.Immunol. 96:122–128, 1994; Wheatcroft et al, Hum. Reprod 12:2617–2622,1997). Expression vectors can be constructed using the human MATER cDNAto produce the recombinant human MATER protein in either bacteria orbaculovirus (as described in Example 8). By affinity purification,unlimited amounts of pure recombinant MATER protein can be produced.

An assay kit could provide the recombinant protein as an antigen andenzyme-conjugated goat anti-human IgG as a second antibody as well asthe enzymatic substrates. Such kits can be used to test if the patientsera contain antibodies against human MATER.

This disclosure provides Mater nucleic acids and proteins, including thehuman Mater molecules described above. The disclosure further providesmethods employing these molecules, including methods to predict and/ordiagnose infertility, reduced fertility, or reproductive failure infemales, as well as treatments for such infertility and reducedfertility, and contraceptives. It will be apparent that the precisedetails of the molecules and methods described may be varied or under-or overexpression, are also contemplated. Such kits will includemodified without departing from the spirit of the described invention.The inventors claim all such modifications and variations that fallwithin the scope and spirit of the claims below.

1. An isolated nucleic acid molecule encoding a human MATER protein comprising the amino acid sequence of SEQ ID NO:
 24. 2. The isolated nucleic acid molecule of claim 1, comprising the nucleotide sequence of SEQ ID NO: 23; or a nucleotide sequence that encodes a MATER protein comprising the amino acid sequence of SEQ ID NO:24, but which varies from SEQ ID NO: 23 by virtue of the degeneracy of the genetic code.
 3. A recombinant nucleic acid molecule comprising a promoter sequence operably linked to the nucleic acid molecule of claim
 1. 4. An isolated cell transformed with the nucleic acid molecule of claim
 1. 5. An isolated cell transformed with the recombinant nucleic acid molecule of claim
 3. 